LONG-TERM MAINTENANCE IN OSMUNDA REGALIS L. IN VITRO CULTURE AND EVALUATION OF ITS GENETIC STABILITY USING SCOT MARKERS

Abstract

Osmunda regalis L. is a species listed in the Red Data Book of the Russian Federation and has the category “Endangered”. About 30 plants of this species are currently preserved in the Sochi National Park. The catastrophic decline in the number of this species in its habitat has put it under the threat of total extinction in the Sochi Black Sea region. The problem of fern reproduction is aggravated by the fact that the spores do not germinate in the soil in wet conditions for a long time and quickly lose viability. Therefore, the FIC SSC RAS is working on the conservation and reproduction of O. regalis by biotechnology methods in in vitro culture . The aim of our research was to study physical factors of deposition under in vitro conditions and selection of informative SCOT-primers to assess the genetic stability of Osmunda regalis L. The object of research was gametophytes of O. regalis. The influence of temperature ( ^13oC, ^21oC , ^23oC) and illumination (800 lx, 1000 lx and 3500 lx) on morphometric indices and percentage of gametophyte viability during long-term cultivation (250 days) in in vitro conditions was shown. It was found that morphometric indices and percentage of gametophyte viability (41.5-43.6 %) decreased with decreasing temperature (13 ^oC) and illumination (800-1000 lx). It was found that morphometric indices and percentage of gametophyte viability (41.5-43.6 %) decreased with decreasing temperature (13 ^oC) and illumination (800-1000 lx). It was revealed that the optimal conditions for long-term cultivation of O. regalis are temperature 21 ^oC and illumination 800-1000 lx, at which the viability of gametophytes corresponded to 73.5-78.2 %. To evaluate the genetic stability of samples from in vitro culture, 36 SCOT primers were tested, among which eight (SCoT1, SCoT2, SCoT9, SCoT10, SCoT21, SCoT22, SCoT24, SCoT34) showed a low level of amplification. Two markers, SCoT19 and SCoT29, were found to generate the highest number of bands. At the same time, 9 more SCoT markers (SCoT3, SCoT8, SCoT13, SCoT14, SCoT15, SCoT16, SCoT18, SCoT20, SCoT32) showed efficiency, which can be used in the assessment of intraspecific diversity of O. regalis. Analysis of the obtained data revealed genetic stability between long-term (5 years) cultivated gametophytes of O. reg alis L. in the in vitro collection of the genebank and mother plants growing in the botanical garden “Friendship Tree” of the FIC SSC RAS.