Germination and Protocorm Formation of Ophrys Sphegodes Mill. – In Vitro Protocol for a Rare Orchid Species

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.