The Studying of Regeneration Ability of Dactylorhiza majalis and Orchis morio in Vitro

Alambets T.M., Dernkiv O.T., Zagulsky M.M.

Institute of Ecology of the Carpathians, National Academy of Sciences ofUkraine,11 Stefanyk Str., Lviv, 290000 Ukraine
Franko Lviv State University,4 Grushevsky Str., Lviv, 290005 Ukraine

Year by year orchid species are disappearing from their natural habitats at an alarming rate, such that there is a real danger of some species becoming extinct in the near future. Many species of Orchidaceae family of the Carpathian flora are need in preservation. At the same time some orchid species (Dactylorhiza majalis. Orchis morio, for example) are valuable herbs. A large scale storage of rare herbs has led to the threat of disappearing of many species of Carpathian flora. The method of plant tissue culture can solve this problem. Growing tissue in vitro and the clonal reproduction of orchid species gives a possibility to study the special features of their individual development and morphogenesis. Also the method of plant tissue culture allows to obtain the biological active substances from the plant tissue mass, which is grown on the aseptic nutrient medium. That is why this method can help to decrease the rate of destroying of rare herbs from their natural habitats.

The chief purpose of our investigation was to find the suitable media and conditions for theobtaining and growing of callus tissue from seedlings of Dactylorhiza majalis (Reihenb.f.) P.F. Hunt et Summerhayes and Orchis morio L. species and for the clonal reproduction of this species. We use the asymbiotic technique for the germination of seeds of D. majalis and O. morio. Before sowing on aseptic nutrient media seeds were soaked in mixture of solutions of gibberelic acid and dymethylsulfoxide. Seeds before sowing treated with 1% H2SO4 and then immediately with 10% CaOCl2 solution. Seeds were surface sterilised in 96% C2H5OH for 3-5 minutes followed by tree washes with sterile distilled water. Subsequently, the seeds were transferred to sterile nutrient media in Petri dishes for asymbiotic germination. The most suitable medium for D. majalis and O. morio seeds germination occurred H-medium (Harvais, 1973) with auxin and kinetin. The seeds were germinated in the dark. First protocorms appeared in 40-50 days and were grown during 30-45 days. On this stage of development protocorms of O. morio were transferred on modified MS (Murashige, Skoog, 1962) medium, which included auxin, kinetin, 2,4-dichlorophenoxyacetic acid, alpha-naphtylphenoxyacetic acid. Protocorms formed callus tissue on this media in 20-30 days. Protocorms of D. majals stayed at H-medium and after 10-15 days first seedlings with roots and shoots appeared. Seedlings were grown at light 2000 lx. In age of 40-50 days segments of seedlings were transferred on modified MS medium. In 25-30 days callus tissue appeared. With the view of clonal micropropagation of D. majalis and O. morio the suitable medium for growing of callus tissue was found.