High-efficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl.

  • P. Mohanty
  • M. C. DasEmail author
  • S. Kumaria
  • P. Tandon
Original Paper


An efficient protocol for cryopreservation of protocorm like bodies (PLBs) of Dendrobium nobile, based on encapsulation–dehydration (ED) and encapsulation–vitrification (EV), was established. In both cryogenic procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix [comprising 3% (w/v) sodium alginate and 0.1 M CaCl2]. Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2% respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. In both the cases (ED and EV), shoots regenerated from cryopreserved PLBs with an intermediary PLB formation. Regenerated shoots were successfully rooted in the medium containing 1.5 mg/l Indole-3 butyric acid. Successful acclimatization of plantlets was obtained in the compost containing brick pieces and charcoal chunks (1:1) + a top layer of moss with a maximum survivability (82%). EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile. Regenerated plantlets showed normal morphology as that of control plants.


Dendrobium nobile Protocorm like bodies Cryopreservation 







Plant vitrification solution 2


Protocorm like bodies


α-Naphthaleneacetic acid


Indole-3 butyric acid



This research was financially supported by the Centre for Advanced studies in Botany, North-Eastern Hill University (NEHU), Shillong, India. The authors are also thankful to SAIF, NEHU for supply of liquid nitrogen.


  1. Aktar S, Nasiruddin KM, Huq H (2007) In vitro root formation in Dendrobium orchid plantlets with IBA. J Agric Rural Dev 5:48–51Google Scholar
  2. Anthony JJJ, Keng CL, Rathinam X, Sinniah UR, Subramaniam S (2010) Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm-like bodies by vitrification. Afr J Biotechnol 9:7063–7070CrossRefGoogle Scholar
  3. Bian HW, Wang JH, Lin WQ, Han N, Zhu MY (2002) 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 159:113–1145CrossRefGoogle Scholar
  4. Chen Y, Wan JH, Huan CN (2001) Germplasm cryopreservation of Dendrobium candidum by vitrification. J Zhejiang Univ (Agric & Life Sci) 27:436–438Google Scholar
  5. Das MC, Kumaria S, Tandon P (2011) Storage and high conversion frequency of encapsulated protocorm-like bodies of Cymbidium devonianum (orchid). J Hortic Sci Biotechnol 86:611–615CrossRefGoogle Scholar
  6. Dohling S, Kumaria S, Tandon P (2008) Optimization of nutrient requirements for asymbiotic seed germination of Dendrobium longicornu Lindl. and D. formosum Roxb. Proc Indian Natl Sci Acad 74:167–171Google Scholar
  7. Engelmann F (1997) In vitro conservation methods. In: Callow CA, Ford-Lloyd BV, Newbury HJ (eds) Biotechnology and plant genetic resources. CAB International, Oxford, pp 119–161Google Scholar
  8. Faria RT, Illg RD (1995) Propagação clonal de híbridos de Dendrobium nobile Lindl. In: Congresso Brasileiro de Floriculturae Plantas Ornamentais, 10. Campinas. Anais. Campinas (ed) SBF, pp 40–41Google Scholar
  9. Giridhar P, Obul RB, Ravishankar GA (2001) Silver nitrate influences in vitro shoot multiplication and root formation in Vanilla planifolia. Androl Curr Sci 81:1166–1170Google Scholar
  10. Gonzalez-Arnao MT, Engelmann F, Urra C, Morenza M, Rios A (2000) Cryopreservation of citrus apices using the encapsulation–dehydration technique. Cryo Lett 19:177–182Google Scholar
  11. Hazubska-Przybył T, Chmielarz P, Michalak M, Bojarczuk K (2010) Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce). Plant Cell Tiss Org Cult 102:35–44CrossRefGoogle Scholar
  12. Hirai D, Sakai A (1999) Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation-vitrification. Plant Cell Rep 19:150–155CrossRefGoogle Scholar
  13. Hong SR, Yin MH, Shao XH, Wang AP, Xu WH (2009) Cryopreservation of embryogenic callus of Dioscorea bulbifera by vitrification. Cryo Lett 30:64–75Google Scholar
  14. Hu SY (1970) Dendrobium in Chinese medicine. Econ Bot 24:165–174CrossRefGoogle Scholar
  15. Hua YM, Rong HS (2010) A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation-vitrification. Plant Cell Tissue Org Cult 102:35–44CrossRefGoogle Scholar
  16. Hynniewta SR, Kumar Y (2008) Herbal remedies among the Khasi traditional healers and village folks in Meghalaya. Ind J Trad Knowl 7:581–586Google Scholar
  17. Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757CrossRefGoogle Scholar
  18. Jitsopakul N, Thammasiri K, Ishikawa K (2007) Cryopreservation of Vanda coerulea protocorms by encapsulation–dehydration method. In: 33rd Congress on Science and Technology of ThailandGoogle Scholar
  19. Khoddamzadeh A, Sinniah U, Kadir MA, Kadzimin S, Mahmood M, Sreeramanan S (2010) Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson. Afr J Biotechnol 9:6632–6639Google Scholar
  20. Khoddamzadeh A, Sinniah U, Lynch P, Kadir MA, Kadzimin S, Mahmood M (2011) Cryopreservation of protocorm-like bodies (PLBs) of Phaleonopsis bellina (Rchb.f) Christenson by encapsulation-dehydration. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-011-9997-4
  21. Kim YS, Hahn EJ, Yeung EC, Paek KY (2003) Lateral root development and saponin accumulation as affected by IBA or NAA in adventitious root cultures of Panax ginseng CA Meyer. In Vitro Cell Dev Biol 39:245–249CrossRefGoogle Scholar
  22. Lurswijidjarus W, Thammasiri K (2004) Cryopreservation of shoot tips of Dendrobium Walter Oumae by encapsulation-dehydration. Sci Asia 30:293–299CrossRefGoogle Scholar
  23. Maneerattanarungroj P, Bunnag S, Monthatong M (2007) In vitro conservation of Cleisostoma areitinum (Rchb.f.) Garay, rare Thai orchid species by an encapsulation-dehydration method. Asian J Plant Sci 6:1235–1240CrossRefGoogle Scholar
  24. Maruyama E, Ishii K, Kinoshita I (1998) Alginate encapsulation technique and cryogenic procedure for long term storage of the tropical forest tree Guazuma crinite Mart. In Vitro cultures. Japan Agric Res Q 32:301–309Google Scholar
  25. Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13:442–446CrossRefPubMedGoogle Scholar
  26. Moges AD, Shibli RA, Karam NS (2004) Cryopreservation of African Violet (Saintpaulia ionantha Wendl.) shoot-tips. In Vitro Cell Dev Biol Plant 40:389–395CrossRefGoogle Scholar
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  28. Nongdam P, Nirmala C, Tewari R (2006) In vitro multiplication of Cymbidium pendulum orchids via embryo culture. Plant Cell Biotech Mol Biol 7:145–150Google Scholar
  29. Padro MDA, Frattarelli A, Sgueglia A, Condello E, Damiano C, Caboni E (2011) Cryopreservation of white mulberry (Morus alba L.) by encapsulation-dehydration and vitrification. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-011-0017-5
  30. Peng-Fei A, Lu LP, Song JJ (2011) Cryopreservation of in vitro-grown shoot-tips of Rabdosia rubescens by encapsulation-dehydration and evaluation of their genetic stability. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-011-0049-x
  31. Pornchuti W, Thammasiri K (2008) Cryopreservation of protocorms of Dendrobium virgineum Rchb.f. Acta Hortic 788:63–68CrossRefGoogle Scholar
  32. Pouzi NZ, Rathinam X, James AJJ, Poobathy R, Subramaniam S (2011) Early investigation on cryopreservation of Dendrobium sonia-28 using encapsulation-dehydration with modified Evan blue assay. Afr J Biotechnol 10:3534–3539Google Scholar
  33. Sharaf SA, Shibli RA, Kasrawi MA, Baghdadi SH (2011) Cryopreservation of wild Shih (Artemisia herba-alba Asso.) shoot tips by encapsulation-dehydration and encapsulation-vitrification. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-011-0054-0
  34. Subramaniam S, Sinniah UR, Khoddamzadeh AA, Periasamy S, James JJ (2011) Fundamental concept of cryopreservation using Dendrobium sonia-17 protocorm-like bodies by encapsulation-dehydration technique. Afr J Biotechnol 10:3902–3907Google Scholar
  35. Suzuki M, Hayakawa Y, Aoki K (1973) Stereochemistry of intermediates in the syntheses of Dendrobium alkaloids. Tetrahedron Lett 4:331–334CrossRefGoogle Scholar
  36. Suzuki M, Ishikawa M, Akihama T (1998) A novel preculture method for the induction of desiccation tolerance in gentian axillary buds for cryopreservation. Plant Sci 135:69–76CrossRefGoogle Scholar
  37. Thammasiri K (2000) Cryopreservation of seeds of a Thai orchid by vitrification. Cryo Lett 21:237–244Google Scholar
  38. Thammasiri K (2008) Cryopreservation of some Thai orchid species. Acta Hortic 788:53–62CrossRefGoogle Scholar
  39. Vacin EF, Went FW (1949) Some pH changes in nutrient solutions. Bot Gaz 110:605–613CrossRefGoogle Scholar
  40. Valentovie P, Luxová M, Kolaroviè L, Ga š paríková O (2006) Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars. Plant Soil Env 52:186–191CrossRefGoogle Scholar
  41. Vendrame WA, Faria RT (2011) Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms. Sci Hortic 128:131–135Google Scholar
  42. Wang QC, Gafny R, Sahar N, Sela I, Mawassi M, Tanne E, Perl A (2002) Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions and subsequent plant regeneration by encapsulation-dehydration. Plant Sci 162:551–558CrossRefGoogle Scholar
  43. Wang Q, Laamanen J, Uosukainen M, Valkonen JPT (2005) Cryopreservation of in vitro-grown shoot-tips of raspberry (Rubus idaeus L.) by encapsulation-vitrification and encapsulation-dehydration. Plant Cell Rep 24:280–288CrossRefPubMedGoogle Scholar
  44. Xue SH, Luo XJ, Wu ZH, Zhang HL, Wang XY (2008) Cold storage and cryopreservation of hairy root cultures of medicinal plant Eruca sativa Mill. Astragalus membranaceus and Gentiana macrophylla Pall. Plant Cell Tissue Organ Cult 92:251–260CrossRefGoogle Scholar
  45. Yin M, Hong S (2009) Cryopreservation of Dendrobium candidum Wall. Ex Lindl. protocorm-like bodies by encapsulation-vitrification. Plant Cell Tissue Organ Cult 98:179–185CrossRefGoogle Scholar
  46. Yin M, Hong S (2010) A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation–vitrification. Plant Cell Tissue Organ Cult 101:349–358CrossRefGoogle Scholar
  47. Zhang YX, Wang JH, Bian H, Zhu MY (2001) Pregrowth-desiccation: a simple and efficient procedure for the cryopreservation of rice (Oryza sativa L.) embryogenic suspension cells. Cryo Lett 22:221–228Google Scholar
  48. Zhao W, Ye Q, Tan X, Jiang H, Li X, Chen K, Kinghorn AD (2001) Three new sesquiterpene glycosides from Dendrobium nobile with immunomodulatory activity. J Nat Prod 64:1196–1200CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Plant Biotechnology Laboratory, Centre for Advanced Studies in BotanyNorth Eastern Hill UniversityShillongIndia

Personalised recommendations