In vitro Propagation and Forest Reestablishment of Cymbidium finlaysonianum Lindl., an Endangered Medicinal Orchid


  • Suphat RITTIRAT Faculty of Science and Technology, Nakhon Si Thammarat Rajabhat University, Nakhon Si Thammarat 80280
  • Sutha KLAOCHEED Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla University, Pattani campus, Pattani 94000
  • Kanchit THAMMASIRI Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400
  • Somporn PRASERTSONGSKUN Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani campus, Pattani 94000



In vitro propagation, Cymbidium finlaysonianum, acclimatization, forest reestablishment, boat orchid


An in vitro plant regeneration protocol was successfully established for Cymbidium finlaysonianum Lindl., an epiphytic endangered medicinal orchid, by culturing seeds from 4-month-old green self-pollinated fruits. Tetrazolium (TZ) viability test indicated a mean embryo viability of 97.80 %. Three asymbiotic orchid seed germination media, namely Knudson C medium (KC), New Dogashima medium (ND), and Vacin and Went medium (VW), were studied to select a suitable medium for seed germination. The highest seed germination percentage was 83.8 % on VW medium. Protocorm-like bodies (PLBs) of C. finlaysonianum Lindl. induced from protocorm segments cultured in VW liquid medium supplemented with 8.84 µM 6-benzylaminopurine (BAP) gave the highest % PLB formation and number of PLBs per explant, at 85.7 and 5.2, respectively. VW medium supplemented with 0.2 % (w/v) activated charcoal (AC) gave the highest number of roots per plantlet and root length, at 6.4 roots and 5.2 cm, respectively. The highest percentage of plantlet survival recorded at 24 months after reestablishment in the forest was 71.6 %.


Download data is not yet available.


Metrics Loading ...


ND Swarts and KW Dixon. Terrestrial orchid conservation in the age of extinction. Ann. Bot. 2009; 104, 543-56.

WM Ferreira and RM Suzuki. O Cultivo in vitro de Orquídeas como Alternativa Para a Preservação de Espécies Nativas Ameaçadas de Extinção. In: MIB Loiola, IG Baseia and JE Lichston (eds.). Atualidades, Desafios e Perspectiva da Botânica no Brasil. Imagem Gráfica, Natal, 2008, p. 67-8.

D Dutra, ME Kane and L Richardson. Asymbiotic seed germination and in vitro seedling development of Cyrtopodium punctatum: A propagation protocol for an endangered Florida native orchid. Plant Cell Tissue Organ. Cult. 2009; 96, 235-43.

RM Suzuki, VC Moreira, M Nakabashi and WM Ferreira. In vitro germination and growth of Hadrolaelia tenebrosa (Rolfe) Chiron & V.P. Castro (Orchidaceae), an endangered species of the Brazilian flora. Hoehnea 2009; 36, 657-66.

T Godo, M Komori, E Nakaoki, T Yukawa and K Miyoshi. Germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cell Dev. Biol. Plant 2010; 46, 323-8.

JJ Wood, RS Beaman, R Repin and JJ Vermeulen. The Orchids of Mount Kinabalu. Vol II. Natural History Publications, Borneo, 2011, p. 1-436.

S Chugh, S Guha and I Usha Rao. Micropropagation of orchids: A review on the potential of different explants. Sci. Hortic. 2009; 22, 507-20.

MM Hossain, M Sharma, JAT da Silva and P Pathak. Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Sci. Hortic. 2010; 123, 479-87.

CR Deb and A Pongener. Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: A multipurpose orchid. J. Plant Biochem. Biotechnol. 2011; 20, 90-5.

JAT da Silva, DTT Giang, MT Chan, AS Norikane, ML Chai, J Chico-Ruiz, S Penna, T Granstrom and M Tanaka. The influence of different carbon sources, photohetero-, photoauto-and photo mixotrophic conditions on protocorm-like body organogenesis and callus formation in thin cell layer culture of hybrid Cymbidium (Orchidaceae). Orchid Sci. Biotechnol. 2007; 1, 15-23.

RB Malabadi, JAT da Silva, K Nataraja and GS Mulgund. Shoot tip transverse thin cell layers and 2, 4-epibrassinolide in the micropropagation of Cymbidium bicolor Lindl. Floricult. Ornamental Biotechnol. 2008; 2, 44-8.

G Mahendran and VN Bai. Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl. Sci. Hortic. 2012; 135, 40-4.

MM Hossain, R Kant, PT Van, B Winarto, SJ Zeng and JAT. da Silva. The application of biotechnology to orchids. Crit. Rev. Plant Sci. 2013; 32, 69-139.

JAT da Silva. Orchids: Advances in tissue culture, genetics, phytochemistry and transgenic biotechnology. Floricult. Ornamental Biotechnol. 2013; 7, 1-52.

JAT da Silva. The role of thin cell layers in regeneration and transformation in orchids. Plant Cell Tissue Organ. Cult. 2013; 113, 149-61.

JAT da Silva and M Tanaka. Embryogenic callus, PLB and TCL paths to regeneration in hybrid Cymbidium (Orchidaceae). J. Plant Growth Regul. 2006; 25, 203-10.

G Lakon. The topographical tetrazolium method for determining the germinating capacity of seeds. Plant Physiol. 1949; 24, 389-94.

L Knudson. A new nutrient solution for germination of orchid seeds. Am. Orchid Soc. Bull. 1946; 15, 214-17.

K Tokuhara and M Mii. Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep. 1993; 13, 7-11.

EF Vacin and FW Went. Some pH changes in nutrient solutions. Bot. Gaz. 1949; 110, 605-17.

F Singh. Differential staining of orchid seeds for viability testing. Am. Orchid Soc. Bull. 1981; 50, 416-8.

HW Pritchard. Determination of orchid seed viability using fluorescein diacetate. Plant Cell Environ. 1985; 8, 727-30.

JJJ Antony, UR Sinniah, CL Keng, R Pobathy, AA Khoddamzadeh and S Subramaniam. Selected potential encapsulation-dehydration parameters on Dendrobium Bobby Messina protocorm-like bodies using TTC analysis. Aus. J. Crop Sci. 2011; 5, 1817-22.

S Subramaniam, UR Sinniah, AA Khoddamzadeh, S Periasamy and JJ James. Fundamental concept of cryopreservation using Dendrobium sonia-17 protocorm-like bodies by encapsulation-dehydration technique. Afr. J. Biotechnol. 2011; 10, 3902-7.

RF JR Galdiano, EGM Lemos, RT Faria and WA Vendrame. Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and Supercool X1000. Sci. Hortic. 2012; 148, 154-60.

RF JR Galdiano, EGM Lemos, RT Faria and WA Vendrame. Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds. Appl. Biochem. Biotechnol. 2014; 172, 2521-9.

H Verleysen, G Samyn, EV Bockstaele and P Debergh. Evaluation of analytical techniques to predict viability after cryopreservation. Plant Cell Tissue Organ. Cult. 2004; 77, 11-21.

RM Suzuki, VC Moreira, R Pescador and WM Ferreira. Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina. In Vitro Cell Dev. Biol. Plant 2012; 48, 500-11.

PJ Kauth, WA Vendrame and ME Kane. In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tissue Organ. Cult. 2006; 85, 91-102.

H Hartman, D Kester, F Davis and R Geneve. Plant Propagation; Principles and Practices. 6th ed. Prentice-hall, New Jersey, 1997, p. 125-44.

Sungkumlong and CR Deb. Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook. Indian J. Exp. Biol. 2008; 46, 243-8.

TR Johnson, SL Stewart, D Dutra, ME Kane and L Richardson. Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae): Preliminary evidence for the symbiotic culture advantage. Plant Cell Tissue Organ. Cult. 2007; 90, 313-23.

G Mahendran, V Muniappan, M Ashwini, T Muthukumar and VN Bai. Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development. Acta Physiol. Plant 2013; 35, 829-40.

D Lauzer, S Renaut, MS Arnaud and D Barabe. In vitro asymbiotic germination, protocorm development and plantlet acclimatization of Aplectrum hyemale (Muhl. ex. Willd.) Torr. (Orchidaceae). J. Torrey Bot. Soc. 2007; 134, 344-8.

RM Suzuki, V Almeida, R Pescador and WM Ferreira. Germinação e crescimento in vitro de Cattleya bicolor Lindley (Orchidaceae). Hoehnea 2010; 37, 731-42.

J Arditti and R Ernst. Micropropagation of Orchid. John Wiley and Sons, New York, 1993.

PJ Kauth, D Dutra, TR Johnson, SL Stewart, ME Kane and W Vendrame. Techniques and Applications of in vitro Orchid Seed Germination. 1st ed. Isleworth, Global Science Books, UK, 2008, p. 375-91.

S Paul, S Kumaria and P Tandon. An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India. AoB Plants 2012; 2012, plr032.

J Arditti and R Ernst. Physiology of Germinating Orchid Seeds. In: J Arditti (ed.). Orchid Biology Reviews and Perspectives III. Cornell University Press, New York, 1984, p. 178-222.

V Raghavan and JG Torrey. Inorganic nitrogen nutrition of the seedling of the orchid Cattleya. Am. J. Bot. 1964; 51, 264-74.

SL Stewart and ME Kane. Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue Organ. Cult. 2006; 86, 147-58.

T Murashige and F Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 1962; 15, 473-97.

A Mohapatra and GR Rout. In vitro micropropagation of Geoderum purpureum R.Br. Indian J. Biotech. 2005; 4, 568-70.

H Sunitibala and R Kishor. Micropropagation of Dendrobium transparens L. from axenic pseudobulb segments. Indian J. Biotech. 2009; 8, 448-52.

D Panwar, K Ram, Harish and NS Shekhawat. In vitro propagation of Eulophia nuda Lindl., an endangered orchid. Sci. Hortic. 2012; 139, 46-52.

AR Roy, RS Patel, VV Patel, S Sajeev and BC Deka. Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid. Sci. Hortic. 2011; 128, 325-31.

S Vyas, S Guha, P Kapoor and U Rao. Micropropagation of Cymbidium Sleeping Nymph through protocorm-like bodies production by thin cell layer culture. Sci. Hortic. 2010; 123, 551-7.

S Rittirat, S Kongruk and S Te-chato. Induction of protocorm-like bodies (PLBs) and plantlet regeneration from wounded protocorms of Phalaenopsis cornu-cervi (Breda) Blume & Rchb. f. J. Agric. Tech. 2012; 8, 2397-407.

JAT da Silva, N Singh and M Tanaka. Priming biotic factors for optimal 479 protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and 480 assessment of cytogenetic stability in regenerated plantlets. Plant Cell Tissue Organ. Cult. 2006; 84, 135-44.

JAT da Silva, T Yam, S Fukai, N Nayak and M Tanaka. Establishment of optimum nutrient media for in vitro propagation of Cymbidium Sw (Orchidaceae) using protocorm-like body segments. Prop. Ornamental Plants 2005; 5, 129-36.

S Seeni and PG Latha. In vitro multiplication and ecorehabilitation of the endangered Blue Vanda. Plant Cell Tissue Organ. Cult. 2000; 61, 1-8.

V Nagaraju and SK Mani. Rapid in vitro propagation of orchid Zygopetalum intermedium. J. Plant Biochem. Biotech. 2005; 14, 27-32.

CR Dev and Temjensangba. In vitro propagation of threatened terrestrial orchid Malaxis khasiana Soland ex. Swartz through immature seed culture. Ind. J. Exp. Biol. 2006; 44, 762-6.

JLS Mayer, GC Stancato and BAD Glória. Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae). Plant Cell Tissue Organ. Cult. 2010; 103, 411-6.

AH Naing, JD Chung, IN Park and KB Lim. Efficient plant regeneration of the endangered medicinal orchid, Coelogyne cristata using protocorm-like bodies. Acta Physiol. Plant 2011; 33, 659-66.

PI Hong, JT Chen and WC Chang. Plant regeneration via protocorm-like body formation and shoot multiplication from seed-derived callus of a maudiae type slipper orchid. Acta Physiol. Plant 2008; 30, 755-9.

CH Huang and JP Chung. Efficient indirect induction of protocorm-like bodies and shoot proliferation using field-grown axillary buds of a Lycaste hybrid. Plant Cell Tissue Organ. Cult. 2010; 106, 31-8.

CY Ng and NM Saleh. In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell Tissue Organ. Cult. 2011; 105, 193-202.

SY Park, HN Murthy and KY Paek. Mass multiplication protocorm like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell Tissue Organ. Cult. 2000; 63, 67-72.

S Rittirat, K Thammasiri and S Te-chato. Effect of media and sucrose concentrations with or without activated charcoal on the plantlet growth of P. cornu-cervi (Breda) Blume & Rchb. f. Int. J. Agric. Tech. 2012; 8, 2077-87.

MJ Pan and J Staden. The use of charcoal in in vitro culture: A review. Plant Growth Regul. 1998; 26, 155-63.

SC Van Winkle and GS Pullman. Achieving desired plant growth regulator levels in liquid plant tissue culture media that include activated carbon. Plant Cell Rep. 2006; 22, 303-11.

JAT da Silva. Impact of paper bridges, activated charcoal, and antioxidants on growth and development of protocorm-like bodies of hybrid Cymbidium. In Vitro Cell Dev. Biol. Plant 2013; 49, 414-20.

G Fridborg, ML Pedersen and T Eriksson. The effect of activated charcoal on tissue cultures: Adsorption of metabolites inhibiting morphogenesis. Physiol. Plant 1978; 43, 104-6.

SJ Nissen and EG Sutter. Stability of IAA and IBA in nutrient medium to several tissue culture procedures. HortScience 1990; 25, 800-2.

L Johansson, E Galleberg and A Gedin. Correlation between activated charcoal, Fe EDTA and other organic media ingredients in cultures of anthers of Anemone canadensis. Physiol. Plant 1990; 80, 243-9.




How to Cite

RITTIRAT, S., KLAOCHEED, S., THAMMASIRI, K., & PRASERTSONGSKUN, S. (2017). In vitro Propagation and Forest Reestablishment of Cymbidium finlaysonianum Lindl., an Endangered Medicinal Orchid. Walailak Journal of Science and Technology (WJST), 15(10), 711–724.



Research Article

Most read articles by the same author(s)