Effects of Coconut Water, Benzylaminopurine, and Naphthalene Acetic Acid on Seed Germination and Rhizome Food Reserve: In Vitro Culture of Eulophia flava (Lindl.) Hook.f.

Main Article Content

Ekarat Vasupen
Weenun Bundithya
Nuttha Potapohn*


ulophia flava (Lindl.) Hook.f. in Thailand has been threatened by agricultural deforestation and city expansion. Efficient seed propagation via tissue culture technique is necessary to enhance the conservation and utilization of this orchid. Thus, the effects of coconut water (CW), benzylaminopurine (BA), and naphthalene acetic acid (NAA) on seed germination and rhizome food reserve were studied. Capsules were collected at 180 days old following hand-pollination. Ten media; MS, MS + 10% CW, MS + 2.69 µM NAA, MS + 5.38 µM NAA, MS + 2.22 µM BA, MS + 4.44 µM BA, MS + 2.69 µM NAA + 2.22 µM BA, MS + 2.69 µM NAA + 4.44 µM BA, MS + 5.38 µM NAA + 2.22 µM BA, and MS + 5.38 µM NAA + 4.44 µM BA, were used for seed germination and seedling development. MS without supplements was found to be suitable for E. flava seed culture, with seed germination of 26.39%, survival of 100%, and seedling development in the post-protocorm stage of 11.50%. After that, seedlings in early stage 4 were selected from MS medium treatment and transferred to five media: MS, 1/2 MS, 1/2 MS macronutrient, MS + 10% CW and MS + 2.69 µM NAA, for formation and food reserves of rhizomes. Four months after being subcultured, seedlings in early stage 4 developed to late stage 4. Rhizome size and cuticle thickness showed no significant differences in all media. Seedlings in 1/2 MS macronutrient showed the highest rhizome food reserve (201.21 mg/g DW of TNC).

Keywords: Eulophia flava; coconut water; plant growth regulators; food reserve; TNC

*Corresponding author: Tel.: (+66) 813876776

                                             E-mail: [email protected]

Article Details

Original Research Articles


Shrivastava, R.J., 2004. Range extension of Eulophia flava (Lindl.) J.D. Hook. (Orchidaceae) in India. Selbyana, 25(1), 23-26.

Rungruang, C. and Sangtongpraow, S., 2001. Population variation of Eulophia flava (Lindl.) Hook.f. in Mae Ping National Park, Lumphun province. Abstracts Research and Thesis 2001: 5th BRT Annual Conference, Biodiversity Research and Training Program, Bangkok, Thailand, October 8-11, 2001, pp. 119.

Kumar, P., Tewari, R. and Rawat, G.S., 2011. Assisted pollination and seed formation for conservation of Eulophia flava (Orchidaceae): a case study from Himalayan foothills, India. Richardiana, 12(1), 25-38.

Rao, B.R.P., Sadasivaiah, B., Prasad, K., Basha, S.K., Miria, A., Khan, A.B. and Babu, M.V.S., 2010. Eulophia flava (Lindley) Hook.f. (Orchidaceae), in Eastern Ghats, India. Indian Journal of Forestry, 33(3), 403-404.

Rasmussen, H.N., 1995. Terrestrial Orchids from Seed to Mycotrophic Plant. New York: Cambridge University.

Johnson, T.R, 2011. Developing a Model of Orchid Seed Germination: In Vitro Studies of the Threatened Florida species Bletia purpurea. Ph.D. University of Florida, USA.

Molnár, Z., Virág, E. and Ördög, V., 2011. Natural substances in tissue culture media of higher plants. Acta Biologica Szegediensis, 55(1), 123-127.

Utami, E.S.W., Purnobasuki, H., Soedarti, T. and Hariyanto, S., 2015. Asymbiotic seed germination and in vitro seedling development of Paphiopedilum liemianum Fowlie, an endangered terrestrial orchid in Northern Sumatra, Indonesia. Journal of Plant Sciences, 10(1), 25-34.

De Pauw, M.A., Remphrey, W.R. and Palmer, C.E., 1995. The cytokinin preference for in vitro germination and protocorm growth of Cypripedium candidum. Annals of Botany, 75, 267-275.

Godo, T., Komori, M., Nakaoki, E., Yukawa, T. and Miyoshi, K., 2010. Germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cellular and Developmental Biology, 46, 323-328.

Stewart, S.L. and Kane, M.E., 2006. Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ Culture, 86, 147-158.

Kaur, S. and Bhutani, K.K., 2011. In vitro propagation of Dendrobium chrysotoxum (Lindl.). Floriculture and Ornamental Biotechnology, 5(1), 50-56.

Chen, Y., Goodale, U.M., Fan, X.L. and Gao, J.Y., 2015. Asymbiotic seed germination and in vitro seedling development of Paphiopedilum spicerianum: an orchid with an extremely small population in China. Global Ecology and Conservation, 3, 367-378.

Smith, D., Paulsen, G.M. and Raguse, C.A., 1964. Extraction of total available carbohydrates from grass and legume tissue. Plant Physiology, 39, 960-962.

Martínez-Vilalta, J., Sala, A., Asensio, D., Galiano, L., Hoch, G., Palacio, S., Piper, F.I. and Lloret, F., 2016. Dynamics of non-structural carbohydrates in terrestrial plants: a global synthesis. Ecological Monographs, 86(4), 495-516.

Zimmerman, J.K. and Whigham, D.F., 1992. Ecological functions of carbohydrates stored in corms of Tipularia discolor (Orchidaceae). Functional Ecology, 6, 575-581.

Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497.

Chaitrakulsup, T., 1981. Seasonal Changes in Total Nitrogen and Total Nonstructural Carbohydrates Contents in Leaves and Stem Apexes of Litchi chinensis Sonn. var. Hong-Huay. MSc. Kasetsart University, Thailand.

Hodge, J.E. and Hofreiter, B.T., 1962. Determination of reducing sugars and carbohydrates. In: R.L. Whistler and M.L. Wolfrom, eds. Methods in Carbohydrates Chemistry. New York: Academic Press, pp. 380-394.

Duncan, D.B., 1955. Multiple range and multiple F test. Biometrics, 11, 1-42.

Kauth, P.J., Dutra, D., Johnson, T.R., Stewart, S.L., Kane, M.E. and Vendrame, W., 2008. Techniques and applications of in vitro orchid seed germination. In: J.A. Teixeira da Silva, ed. Floriculture, Ornamental and Plant Biotechnology. Volume V. Middlesex: Global Science Books, pp. 375-390.

Wang, Y., Li, Z., Huang, L. and Su, J., 2013. In vitro mass scale propagation of wild Cymbidium lowianum with a rare and endangered plant. American Journal of Plant Sciences, 4, 1500-1507.

Bustam, B.M., Dixon, K.W. and Bunn, E., 2014. In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination. Botanical Journal of the Linnean Society, 176, 556-566.

Shekarriz, P., Kafi, M., Deilamy, S.D. and Mirmasoumi, M., 2014. Coconut water and peptone improve seed germination and protocorm like body formation of hybrid Phalaenopsis. Agriculture Science Developments, 3(10), 317-322.

Fernández, V., Guzmán-Delgado, P., Graça, J., Santos, S. and Gil, L., 2016. Cuticle structure in relation to chemical composition: re-assessing the prevailing model. Frontiers in Plant Science, 7, DOI: 10.3389/fpls.2016.00427.

Yeats, T.H. and Rose, J.K.C., 2013. The formation and function of plant cuticles. Plant Physiology, 163, 5-20.

Domínguez, E., Heredia-Guerrero, J.A. and Heredia, A., 2011. The biophysical design of plant cuticles: an overview. New Phytologist, 189, 938-949.

Thangavelu, M. and Muthu, S., 2017. Vegetative anatomical adaptations of Epidendrum radicans (Epidendroideae, Orchidaceae) to epiphytic conditions of growth. Modern Phytomorphology, 11, 117-130.

Muthukumar, T. and Shenbagam, M., 2018. Vegetative anatomy of the orchid Bulbophyllum sterile (orchidaceae: epidendroideae). Lankesteriana, 18(1), 13-22.

Almeida, V.O., Carneiro, R.V., Carvalho, M.A.M., Figueiredo-Ribeiro, R.C.L. and Moraes, M.G., 2015. Diversity of non-structural carbohydrates in the underground organs of five Iridaceae species from the Cerrado (Brazil). South African Journal of Botany, 96, 105-111.

Anuchai, J. and Sasiangdee, A., 2020. Effect of growth regulator on shoot induction from protocorm of Dendrobium Anna. International Journal of Agricultural Technology, 16(6), 1319-1330.

Chidburee, A., Suwanthada, C., Bundithya, W., Ohyama, T. and Ruamrungsri, S., 2007. Effect of day length on growth, photosynthesis and food reserves in Curcuma alismatifolia Gagnep. Journal of Agriculture, 23(2), 105-113.

Changjeraja, R. and Intanon, S., 2016. Differences in day and night temperature on growth and non-structural carbohydrate of Eurycle amboinensis Lindl. Songklanakarin Journal of Plant Science, 3(suppl. II), 64-69.