Some techniques in micropropagation and breeding of Paphiopedilum spp.


  • Hoang Thanh Tung Tay Nguyen Institute for Scientific Research, VAST, 116 Xo Viet Nghe Tinh, Da Lat, Lam Dong, Viet Nam
  • Vu Quoc Luan Tay Nguyen Institute for Scientific Research, VAST, 116 Xo Viet Nghe Tinh, Da Lat, Lam Dong, Viet Nam
  • Duong Tan Nhut Tay Nguyen Institute for Scientific Research, VAST, 116 Xo Viet Nghe Tinh, Da Lat, Lam Dong, Viet Nam



in vitro propagation, Paphiopedilum, protocol


Paphiopedilum orchids are one of the most popular and rare orchid genera sold and exhibited as pot plants and cut flowers. Their wild populations are under the threat of extinction as a result of over-collection and loss of suitable habitats. Reduction in their commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection, to attempt at meeting commercial needs and to re-establish these threatened orchid species back into the wild. Although they are commercially propagated via seed germination in vitro, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This paper aims to provide the most important techniques on Paphiopedilum propagation mainly including plant, cell, tissue and organ culture techniques applied to in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols from ex vitro-derived explants of mature plants.


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. Ng C. Y. and Saleh N. M. - In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell Tiss Org Cult 105 (2011) 193-202.

. Ng C. Y., Saleh . M. and Zaman F. Q. - In vitro multiplication of the rare and endangered slipper orchid, Paphiopedilum rothschildianum (Orchidaceae). Afr J Biotechnol 9 (2010) 2062-2068.

. Lee Y. I., Yeung E. C., Lee N. and Chung M. C. - Embryo development in the lady's slipper orchid, Paphiopedilum delenatii, with emphasis on the ultrastructure of the suspensor. Ann Bot 98 (2006) 1311-1319.

. Knudson L. - La germinación no simbiótica de las semillas de orquideas. Bol Real Soc Española Hist Nat 21 (1921) 250-260.

. Kauth P. J., Dutra D., Johnson T. R., Stewart S. L., Kane M. E. and Vendrame W. - Techniques and applications of in vitro orchid seed germination. In: da Silva J. A. T. (ed.) Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Edn, Vol V), Global Science Books, Isleworth, UK, 2008, pp. 375-391.

. da Silva J. A. T. - Orchids: Advances in tissue culture, genetics, phytochemistry and transgenic biotechnology. Floricult Orn Biotech 7 (2013) 1-52.

. Pierik R. L. M., Sprenkels P. A., Vanderharst B. and Vandermeys Q. G. - Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz in vitro. Sci Hort 34 (1988) 139-153.

. Chen Z. L., Ye X. L., Liang C. Y. and Duan J. - Seed germination in vitro of Paphiopedilum armeniacum and P. micranthum. Acta Hortic Sin 31 (2004b) 540-542.

. Ding C. C., Wu H. and Liu F. Y. - Factors affecting the germination of Paphiopedilum armeniacum. Acta Bot Yunnanica 26 (2004) 673-677.

. Nhut D. T., Trang P. T. T., Vu N. H., Thuy D. T. T., Khiem D. V., Binh N. V. and Van K. T. T. - A wounding method and liquid culture in Paphiopedilum delenatii propagation. Prop Orna Plants 5 (2005) 158-163.

. Long B., Niemiera A. X., Cheng Z. Y. and Long C. L. - In vitro propagation of four threatened Paphiopedilum species (Orchidaceae). Plant Cell Tiss Org Cult 101 (2010) 151-162.

. Arditti J. - Micropropagation of Orchids, 2nd edn. Blackwell Publishing Ltd, Maiden, MA, USA, 2008.

. Stewart J. and Button J. - Tissue culture studies in Paphiopedilum. Amer Orch Soc Bull 35 (1975) 88-95.

. Huang L. C. - A procedure for asexual multiplication of Paphiopedilum in vitro. Am Orchid Soc Bul 57 (1988) 274-278.

. Liao Y. J., Tsai Y. C., Sun Y. W., Lin R. S. and Wu F. S. - In vitro shoot induction and plant regeneration from flower buds in Paphiopedilum orchids. In Vitro Cell Dev Biol – Plant 47 (2001) 702-709.

. Bubeck S. K. - A study of Paphiopedilum meristem culture. Ph.D. Thesis, Rutgers University. University Microfilm International, Ann Arbor, Michigan, The USA, (1973), pp. 109.

. Morel G. - Clonal multiplication of orchid. In: The Orchid. Scientific Studies, ed by Withner C. and Wiley . L. J., New York, The USA, (1974)

. Chugh S., Guha S. and Rao I. U. - Micropropagation of orchids: A review on the potential of different explants. Sci Hortic 122 (2009) 507-520.

. Hossain M. M., Kant R., Van P. T., Winarto B., Zeng S. J. and da Silva J. A. T. - The application of biotechnology to orchids. Crit Rev Plant Sci 32 (2013) 69-139.

. Murashige T. and Skoog F. - A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Plant Physiol 1(1962) 473-497.

. Huang L. C., Lin C. J., Kuo C. I., Huang B. L. and Murashige T. - Paphiopedilum cloning in vitro. Sci Hort 91 (2001) 111-121.

. Nhut D. T., Thuy D. T. T., Don N. T., Luan V. Q., Hai N. T., Van K. T. T. and Chinnappa C. C. - In vitro stem elongation of Paphiopedilum delenatii Guillaumin and shoot regeneration via stem node culture. Prop Orna Plants 7 (2007) 29-36.

. Lin Y. H., Chang C. and Chang W. C. - Plant regeneration from callus culture of a Paphiopedilum hybrid. Plant Cell Tiss Org Cult 62 (2000) 21-25.

. Zeng S. J., Wang J., Wu K. L., da Silva J. A. T., Zhang J. X. and Duan J. - In vitro propagation of Paphiopedilum hangianum Perner & Gruss. Sci Hort 151 (2013) 147-156.

. Chen T. Y., Chen J. T. and Chang W. C. - Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids. Plant Cell Tiss Org Cult 76 (2004a) 11-15.

. Chen T. Y., Chen J. T. and Chang W. C. - Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids. In Vitro Cell Dev Biol – Plant 38 (2002) 595-597.

. Hong P. I., Chen J. T. and Chang W. C. - Plant regeneration via protocorm-like body formation and shoot multiplication from seed derived callus of a Maudiae type slipper orchid. Acta Physiol Plant 30 (2008) 755-759.

. Luan V. Q., Cuong L. K., Tung H. T., Hien V. T., Hieu T. and Nhut D. T. - Effects of shoot tip removal, wounding manipulation, and plant growth regulators on shoot regeneration and plantlet development in Paphiopedilum species. Sci Hortic 256 (2019).

. Luan L. Q., Uyen N. H. P. and Ha V. T. T. - In vitro mutation breeding of Paphiopedilum by ionization radiation. Sci Hort 144 (2012) 1-9.

. Sun Y. W., Liao Y. J., Huang Y. S., Chang Y. S. and Sung J. M. - Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids. Sci Hort 127 (2011) 405-410.

. Lee Y., Chang F. and Chung M. - Chromosome pairing affinities in interspecific hybrids reflect phylogenetic distances among lady's slipper orchids (Paphiopedilum). Ann. Bot. 108 (2011) 113-121.

. Chung S. Y. and Choi S. H. - Genetic variability and relationships among interspecific hybrid cultivars and parental species of Paphiopedilum via ribosomal DNA sequence analysis. Plant Syst Evol 298 (2012) 1897-1907.

. Chen L. J., Liu K. W., Xiao X. J., Tsai W. C., Hsiao Y. Y., Huang J. and Liu Z. J. - The anther steps onto the stigma for self-fertilization in a slipper orchid. PLoS ONE 7 (2012) e37478.

. Huy N. P., Luan V. Q., Cuong L. K., Nam N. B., Tung H. T., Hien V. T., Dung T. L., Paek K. Y. and Nhut T. D. - Strategies for the regeneration of Paphiopedilum callosum through internode tissue cultures using dark light cycles. HortSci 54 (2019a) 920-925.

. Huy N. P., Tam D. T. T., Luan V. Q., Tung H. T., Hien V. T., Ngan H. T. M. , Duy P. N. and Nhut D. T. - In vitro polyploid induction of Paphiopedilum villosum using colchicine. Sci Hortic 252 (2019b) 283-290.




How to Cite

H. T. Tung, V. Q. Luan, and D. T. Nhut, “Some techniques in micropropagation and breeding of Paphiopedilum spp”., Vietnam J. Sci. Technol., vol. 58, no. 4, pp. 393–401, Jul. 2020.



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