Ophrys sphegodes Mill. is a wild orchid species which is threatened and protected due to its pollination biology, small seed and habitat destruction. The aim of this study was to establish asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media Knudson C (KC) and Malmgren (MM), supplemented with organic additives (peptone (PE), L-glutamin (A)e, folic acid, casein hydrolysate (CA)) added separately and control media KC--C and MM--C were used in the present research. All the nutrition media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to 5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. The seeds were examined under two illumination conditions, 0/24 light/dark (L/D) and 16/8 L/D. The presented results indicate a huge influence of illumination and nutrition media on the seed germination and protocorm formation. The seed germination was overall significantly more successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). Protocorm, rhizoids and shoot formation were achieved only on the seeds cultured on MM medium, while the KC medium caused only swelling of the embryo. Organic additives had positive influence on the germination rate. According to the obtained results, the best germination rate and seedling development were achieved on MM-PE media, cultured in dark. The presented procedure accelerates the germination period and can provide a large number of plants in a relatively short period of time so it can be used for conservation programs and mass production protocol.
ARDITTI, J., (1967): Factors affecting the germination of orchid seed. The Botanical Review 33: 1-97.
BOZDEMIR, H., CIG, A., TURKOGLU N. (2018): Effects of different concentrations of carbohydrate form on Orchis Sancta L. propagation in vitro. Applied ecology and environmental research. 16(4):4849-4864.
CHEN, Y., GOODALE, U., FAN, H.L., 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.
DAVY, A.J., HUTCHINGS, M.J., ROBERTS, D.L, ROBBIRT, K.M., (2014): Potential Disruption of Pollination in a Sexually Deceptive Orchid by Climatic Change. Current Biology 24: 2845–2849.
GODO, T., KOMORI, M., NAKAOKI, YUKAWA, T., MIYOSHY, 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-Plant 46: 323-328.
GUPTA, A., (2016): Asymbiotic Seed Germination in Orchids: Role of Organic Additives. Interantional Advanced Research in Science. Engineering and Technology 3(5): 143-147.
HUTCHINGS, M.J., (1989): Population biology and conservation of Ophrys sphegodes, Modern methods in orchid conservation: the role of physiology, ecology and management. Cambridge University press, 101-115.
KITSAKI, C.K., ZYGOURAKI, S., ZIOBORA, M., KINTZIOS, S. 2004. In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae). Plant. Cell. Rep. 23: 284–290. KNUDSON, L., (1922): Non-symbiotic germination of orchid seeds. Bot. Gaz. 73: 1–25.
KNUDSON, L., (1946): A new nutrient solution for the germination of orchid seed. American Orchid Society Bulletin 15, 214-217.
MALMGREN, C., (1996): Orchid propagation. Theory and practice. In: Allen C (Eds.) North American native terrestrial orchids. Propagation and production. North Am Native Terrestrial Orchid Conf Proc. C. Allen, Germantown, Md. pp. 63–71.
MARIĆ, M.M., (1995): Plant tissue culture. Publishing house Draganić, 77-82.
PIERCE, S., GUIDI, V., FERRARIO, A., CERIANI, R., LABRA, M., VAGGE, I., CERABOLINI B. (2015): Pea seeds extracts stimulate germination of the terrestrial orchid Ophrys apifera Huds. during a habitat restoration project.
PONERT, J., FIGURA, T., VOSOLSOBE, S., LIPAVSKA, H., VOHNIK, M., JERSAKOVA J. (2013): Asymbiotic germination of mature seeds and protocorm development of Pseudorchis albida (Orchidaceae) are inhabited by nitrates even at extremely low concentrations, Botany 91, 662-670.
PONERT, J., VOSOLSOBE, S., KMECOVA, K., LIPAVSKA, H., (2011): European orchid cultivation – From seed to mature plant. European Journal of Environmental Sciences 1(2): 95-107.
RASMUSSEN, H.N., (1995): Terrestrial orchids, from seed to mycotrophic plant. Cambridge University Press, Great Britain.
SGARBI, E., GRIMAUDO, M., DEL PRETE, C., (2009): In vitro asymbiotic germination and seedling development of Limodorum abortivum (Orchidaceae). Plant Biosystems 143, 114–119.
STEWART, S., KANE, M., (2006): Asymbiotic seed germination and in vitro seedling development of Habanaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ Culture 86: 147-158.
VAN WAES, J.M,, DEBERGH, P.C. (1986): In vitro germination of some Western European orchids. Physiolia Plantarum 67:253–261