PRESERVATION OF THE BULGARIAN ENDEMIC VERBASCUM DAVIDOFFII (SCROPHULARIACEAE) BY MEANS OF IN VITRO PROPAGATION
MARINA STANILOVA*, STOYAN STOYANOV, BORYANA SIDZHIMOVA, BORYANKA TRAYKOVA
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Department of Plant and Fungal Diversity and Resources, Akad. G. Bonchev Str., bl.23, 1113 Sofia, Bulgaria
*Corresponding author: marina.stanilova@gmail.com
Keywords: plant biotechnology, seed germination, endangered plants, reintroduction
Abstract: Verbascum davidoffii Murb. (Scrophulariaceae) is one of the rarest plant species in the Bulgarian flora. It is a Bulgarian endemic, protected by the national Biodiversity Act (2002), included in the Red List of vascular plants in Bulgaria and in the Red Data Book of Bulgaria with conservation status “Critically Endangered”. The populations of this biennial herbaceous plant consist of sparse individuals and are severely fragmented; all known localities are within Pirin National Park and in a site of the European ecological network Natura 2000 in Bulgaria, between the valleys of Banderitsa River and Razlozhki Suhodol, at an altitude of 1800 up to 2300 m a.s.l. The distribution of V. davidoffii is limited due to its peculiar biology related to specific ecological requirements and low reproductive capability, as well as to the anthropogenic pressure: forest felling, destruction and pollution of the habitats because of tourism and infrastructure development in the region.
In order to preserve this species of conservation value, ex situ and in situ activities have been designed concerning elaboration of a specific protocol for in vitro propagation, followed by establishment of an ex situ collection, and strengthening of the wild populations. As a first step, seed germination has been studied. Seeds were gathered in August 2015 from the locality close to the “Banderitsa” rest-house and were successfully disinfected through a standard laboratory procedure. Seed germination was poor on the basal MS medium: only 1 seedling was obtained from 100 seeds for a period of 6 weeks; and no seed germinated on MS medium supplemented with 1 mg/l Kin. The stimulation of the process by seeds soaking in 0,35 % solution of gibberellic acid for 22 hours increased the germination rate up to 61 % and 18 % for the two media, respectively. The effect of the gibberellic acid was strong even if applied for only 2 h, and the concentration of kinetin was better when supplemented in 10-fold less concentration. Seed stratification with low temperature at 6°C for a month prior to cultivation had additional effect on germination which depended on the presence of kinetin in the medium. In vitro seedlings with several leaves and roots were potted in soil substrate of soil mixture, sand and coconut fiber (2:1:1), and easily ex vitro adapted into the ambience of the laboratory phytotron, under controlled temperature, light, and humidity variations.