Efficient Adventitious Shoot Regeneration from Shoot Tip Culture of Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple), a Rare Thai Orchid Species


  • Sasikarn PRASONGSOM Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400
  • Kanchit THAMMASIRI Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400
  • Ngarmnij CHUENBOONNGARM Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400


Rhynchostylis gigantea, shoot tip culture, plant growth regulators


Shoot tip culture was applied to Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple) to increase the number of orchids that have a low population in natural habitats. The effects of sucrose concentrations, plant growth regulators (PGRs), and activated charcoal (AC) in different culture media on protocorm-like body (PLB) induction, multiple shoots, root formation, and ploidy level stability were investigated. The effects of various concentrations of sucrose (0 - 20 g L-1) incorporated in 5 liquid media; Vacin and Went, 1949 medium (VW), VW medium supplemented with 150 ml L-1 coconut water (modified VW), Schenk & Hildebrandt, 1976 medium (SH), Murashige and Skoog, 1962 medium (MS), and half strength MS medium (1/2 MS) were studied. The highest PLBs (7.9 pieces) and survival (92.6 %) were observed in modified VW liquid medium supplemented with 10 g L-1 sucrose without plant growth regulators after 8 weeks of culture. Shoot and root induction were observed in SH agar medium supplemented with PGRs. The supplementation of 0.1 mg L-1 TDZ on SH agar medium induced the highest shoot formation at 73.3 shoots per explant, while in SH agar medium supplemented with NAA induced root formation, the highest root formation was at 4.2 mm. After 12 weeks of culture, SH agar medium showed the release of phenolic compounds. AC was used to inhibit phenolic exudates and to induce root formation in SH agar medium. The combination of 1 g L-1 AC and 1.0 g L-1 NAA showed an optimum root formation at 51 % and rooting from a shoot at 2.3 roots per shoot. A similar pattern of ploidy level was observed by flow cytometry from the leaves of both stock plantlets and regenerated plantlets. There was no difference in the morphology of stock plantlets and regenerated plantlets.


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Author Biography

Sasikarn PRASONGSOM, Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400

Department of Plant Science, Associate Professor


Kew (Royal Botanic Garden), World checklist of selected plant families, Available at: http://apps.kew.org/wcsp/infamilie.do, accessed April 2015.

K Thammasiri. Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima Lindl.) by vitrification. Cryo Lett. 2000; 21, 237-44.

TE Soon. Orchids of Asia. Times books international, Singapore. 1980, p. 143-55.

GM Morel. Producing virus-free cymbidium orchids. Am. Orchid Soc. Bull. 1960; 29, 495-7.

P Pornpienpakdee, R Singhasurasak, P Chaiyasap, R Pichyangkura, R Bunjongrat, S Chadchawan and P Limpanavech. Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan. Sci. Hortic. 2010; 124, 490-9.

P Mohanty, S Paul, MC Das, S Kumaria and P Tandon. A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India. AoB Plants 2012; 2012, pls023.

NV Ket, EJ Hahn, SY Park, D Chakrabarty and KY Peak. Micropropagation of an endangered orchid Anoectochilus formosanus. Plant Biol. 2004; 48, 339-44.

P Lakshmanan, CS Loh and CJ Goh. An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Rep. 1995; 14, 510-4.

B Ravindra, S Gangadhar and K Nataraja. Efficient regeneration of Vanda coerulea, an endangered orchid using Thidiazuron Plant Cell Tissue Organ. Cult. 2004; 76, 289-93.

MO Ialam and S Ichihashi. Effects of sucrose, maltose and sorbitol on callus growth and plantlets regeneration in Phalaenopsis, Doritaenopsis and Neofinetia. J. Jpn. Soc. Hortic. Sci. 1999; 68, 1124-31.

M Yaseen, T Ahmad, G Sablok, A Stamdardi and IA Itafiz. Review: Role of carbon sources for in vitro plant growth and development. Mol. Biol. Rep. 2013; 40, 2837-49.

Y Chen and C Piluek. Effects of thidiazuron and N6- benzylaminopurine on shoot regeneration of Phalaenopsis. Plant Growth Regul. 1995; 16, 99-101.

M Mukarlina, A Listiawati and S Mulyani. The effects of coconut water and naphthalene acetic acid (NAA) application on the in vitro growth of Paraphalaenopsis serpentilingua from West Kalimantan. BioScience 2010; 2, 62-6.

S Asghar, T Ahmad, IA Hafiz and M Yaseen. In vitro propagation of orchid (Dendrobium nobile) var. Emma white. Afr. J. Biotechnol. 2011; 10, 3097-103.

A Husen and M Pal. Metabolic changes during adventitious root primordium development in Tectona grandis Linn. f. (teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment. New Forest. 2007; 33, 309-23.

NR Nayak, S Sahoo, S Patnaik and SP Rath. Establishment of thin cross section (TCS) culture method for rapid micropropagation of Cymbidium aloifolium (L.) Sw. and Dendrobium nobile Lindl. (Orchidaceae). Sci. Hort. 2002; 94, 107-16.

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.

R Ernst. Effect of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant Cell Tissue Organ. Cult. 1994; 39, 273-85.

N Jitsopakul, K Thammasiri and K Iashikawa. Efficient adventitious shoot regeneration from shoot tip culture of Vanda coerulea, a Thai orchid. Sci. Asia 2013; 39, 449-55.

SK Bhadra and MM Hossain. In vitro germination and micropropagation of Geodorum densiflorum (Lam.) Schltr., an endangered orchid species. Plant Tissue Cult. 2003; 13, 165-71.

MK Cheruvathur, J Abraham, B Mani and TD Thomas. Adventitious shoot induction from cultured intermodal explants of Malaxis acuminate D. Don, a valuable terrestrial medicinal orchid. Plant Cell Tissue Organ. Cult. 2010; 101, 163-70.

CP Eliane, MG Leticia, AMG Maria, AM Claudete and FPSM. Activated charcoal and graphite for the micropropagation of Cattleya bicolor Lindl. and a orchid double hybrid ‘BLC Pastoral Innocence’. Acta Sci. Agron. 2012; 34, 157-61.

WR Clarindo, CR Carvalho, MAC Mendonca. Ploidy instability in long-term in vitro cultures of Coffea Arabica L. monitored by flow cytometry. Plant Growth Regul. 2012; 68, 533-8.

ZY Li and L Xu. In vitro propagation of white-flower mutant of Rhynchostylis gigantea (Lindl.) Ridl. through immature seed-derived protocorm-like bodies. J. Hort. Forest. 2009; 1, 93-7.

TD Thomas and A Micheal. High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid. Plant Biotechnol. Rep. 2007; 1, 243-9.

M Vajarabhaya and T Vajarabhaya. Culture of Rhynchostylis gigantea a monopodial orchid. Am. Orchid Soc. Bull. 1970; 39, 907-10.

SP Vij, P Pathak and M Sharma. On the regeneration potential of Rhynchostylis retusa root segments: A study in vitro. J. Orchid Soc. India 1987; 1, 71-84.

B Bui Van Le, NTH Phuong, LTA Hong and KTT Van. High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers. Plant Growth Regul. 1999; 28, 179-85.

S Te-chato, S Kongruk and W Khaimuk. Micropropagation of Chang Daeng (Rhynchostylis gigantea var. rhubrum Sagarik) by embryogenic callus. J. Agric. Sci. Tech. 2010; 6, 589-97.

SJ Ochatt. Flow cytometry in plant breeding. Cytometry A 2008; 73, 581-98.

J Greilhuber and J Dolzel. 2C or not 2C: A closer look at cell nucli and their DNA content. Chromosoma 2009; 118, 391-400.

J Arditti and R Ernst. Orchid Biology: Review and prospective-III. In: J Arditi (ed.). Physiology of Germinating Orchid Seeds. Cornell University Press, New York, 1984, p. 177-222.

PJ Kauth, D Dutra, TR Johnson, SL Stewart, W Vendrame and ME Kane. Technique and applications of in vitro orchid seed germination. 1st ed. Isleworth, Global Science Books, UK, 2008, p. 375-91.

A Indrianto, BE Hererle and A Touaev. Assessment of various stress and carbohydrates for their effects on the induction of embryogenesis isolated wheat microspores. Plant Sci. 1990; 143, 71-9.

MO Islam, S Ichihashi and S Matsui. Control of growth and development of protocorm-like body derived from callus by carbon sources in Phalaenopsis. Plant Biotechnol. 1998; 15, 183-7.

MO Islam and S Ichihashi. Effects of sucrose, maltose and sorbitol on callus growth and plantlet regeneration in Phalaenopsis, Doritaenopsis and Neofinetia. J. Jpn. Soc. Sci. 1999; 68, 11.

J Arditti and R Emst. Micropropagation of Orchids. John Wiley and Sons, New York, 1993.

MC Mok, WS Mok, DJ Armostong, K Shudo, YI Sogai and T Okamoto. Cytokinin activity of N-phenyl-N-1,2,3-thiadiazol-5ylurea (thidiazuron). Phytochemistry 1982; 21, 1509-611.

JT Chen and WC Chang. Effect of auxins and cytokinins on direct somatic embryogenesis of leaf explants of Oncidium ‘Gower Ramsey’. Plant Growth Regul. 2001; 34, 229-32.

NR Nayak, S Patnaik and RP Rath. Direct shoot regeneration form foliar explants of epiphytic orchid Acampae praemorsa (Roxb.) Blatter and McCann. Plant Cell Rep. 1997; 16, 583-96.

LR Chen, JT Chen and WC Chang. Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid Epidendrum redicans. In Vitro Cell Dev. Biol. Plant. 2002; 38, 441-5.

RB Malabidi, GS Mulgund and K Nataraja. Efficient regeneration of Vanda coerulea, an endangered orchid using thidiazuron. Plant Cell Tissue Organ. Cult. 2004; 76, 289-93.

J Roy, S Naha, M Majumdar and N Banerjee. Direct and callus mediated protocorm-like body induction from shoot-tips of Dendrobium chrysotoxum Lindl. (Orchidaceae). Plant Cell Tissue Organ. Cult. 2007; 90, 31-43.

S Samarfard and MA Kadir. In vitro propagation and detection of somaclonal variation in Phalaenopsis gigantea as affected chitosan and thidiazuron. HortScience 2014; 49, 82-8.

TD Thomas. The role of activated charcoal in plant tissue culture. Biotechnol. Adv. 2008; 26, 618-31.

K Sopalun, K Thammasiri and K Ishikawa. Micropropagation of the Thai orchid Grammatophyllum speciosum blume. Plant Cell Tissue Organ. Cult. 2010; 101, 119-26.




How to Cite

PRASONGSOM, S., THAMMASIRI, K., & CHUENBOONNGARM, N. (2016). Efficient Adventitious Shoot Regeneration from Shoot Tip Culture of Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple), a Rare Thai Orchid Species. Walailak Journal of Science and Technology (WJST), 13(9), 757–767. Retrieved from https://wjst.wu.ac.th/index.php/wjst/article/view/1921

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