CONSERVATION OF THE PROTECTED RESURECTION SPECIES RAMONDA SERBICA PAN.

HABITAT MONTANA DISTRICT, BULGARIA AS IN VITRO PLANTS THROUGH A MODIFIED MICROPROPAGATION SYSTEM
S. Dontcheva, E. Daskalova, G. Yahubyan, I Denev, I. Minkov, V. Toneva University of Plovdiv Paisii Hilendarski, Plovdiv, Bulgaria Correspondence to: Evelina Daskalova E-mail: eve_das@uni-plovdiv.bg

ABSTRACT
Ramonda serbica Pan. is a rare species, Balkan endemic and tertiary relict, included in the European list of rare, in danger of extinction and endemic plants. It is also included in the Bulgarian list of endangered plants. In Bulgaria, this species has not been cultivated in vitro with the aim of conservation and investigation of the natural population. For this purpose, at the Plant Biotechnology laboratory at the University of Plovdiv we started the establishment of a live collection of in vitro Ramonda serbica Pan. This is accomplished through an in vitro system for regeneration and propagation, modified by our research group. The live collection of in vitro Ramonda serbica plants will be a donor for conservation and reintroduction of adapted in vitro plants in their natural endangered habitats and also for physiological studies of drough tolerance, and multidisciplinary comparative analyses. Keywords: in vitro microplants, modified in vitro system, Ramonda serbica Pan. Bulgaria, resurection plants The general research plan is shown on Fig.1.
Expeditions to the habitat (near Prevala villade, Montana)

Introduction
In recent years, the resurrection plants from Gesneriaceae family have become important models for studying drought tolerance (5, 6, 9) , photosynthetic abilities during transition from anabiosis (3, 4) and other specialized and multidisciplinary studies. On the territory of Bulgaria, Ramonda serbica Pan. was discovered in 1937 (1). It is a rare species; Balkan endemic and Tertiary relict, included in the European list of rare, in danger of extinction and endemic plants (2). With respect of the conservation status of the species, we propose as a conservation method the establishment of an in vitro live collection. This is the first application of this method for the Gesneriaceae family. For this purpose, we have developed at the Plant biotechnology laboratory of the University of Plovdiv a modified in vitro system using as a starting material seeds of Ramonda serbica Pan, habitat Prevala, Montana district, Bulgaria. The system will be an integral part of a project recently funded by Bulgarian Ministry of education and science for Sustained management of biodiversity of the Gesneriaceae family.
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Seed collection

Plant Tissue Cultures Laboratory - Dry sterilization of seeds (applied for a first time for Gesneriaceae) - Micropropagation Live collection - Rooting in vitro plant

Ecology sustained management of the biodiversity - reintroduction Fig.1. Conservation of the protected resurection species Ramonda serbica pan. habitat Montana district, Bulgaria as in vitro plants through a modified micropropagation system.

Materials and methods

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stored at dry and airy place at temperature near 25C. II. Germination test The seed capsules were removed, and the very fine seeds (under 0.5 mm in diameter) were then passed through fine sieves. In order to test the viability of the seeds in vivo, they were put on Petri dishes with moist filter paper and stored at 23 until a plantlet with two green leaves appears. III. Modified in vitro micropropagation system We applied a direct organogenesis method, which included the following stages: 1. Dry Sterilization. The seeds were put into eppendorph tubes and were sterilized in an exicator on the fume of sodium hypochloride and HCl. 2. Germination. Sterilized seeds were put in vitro in vials with nutrient medium GM without sugar and hormones, according the protocol of Toth et al (7). At the end of this stage, a normal organogenesis of individual microplants was observed 3. Micropropagation. The Ramonda serbica Pan microplants from the previous stage were transferred on nutrient media RA, described in (7) including combination of phytohormones cytokinin/auxin (6benzyladenine, BAP and indole-3-acetic acid, IAA, 0.1mg/l each), pH 5,8. the medium was supplemented with an antioxidant - 200mg/l filter sterilised glutathione and the pH was stabilized with K-phosphate buffer. 4. Rooting. The growing microplants were transferred on RA medium but only a phytohormone from the auxin group (IAA, 0,2mg/l) was added. At the end of this stage, the microplants formed a stabile root system. 5. Adaptation. The rooted plants were planted in vivo in a turf-perlite mixture 1:1 (v/v) and they were grown under controlled air humidity and temperature in a growth camera for a period of three weeks. At the stages 2, 3 and 4, the microplants were grown in vitro in growth cameras with controlled temperature 21 1, at light regime 16 h light (day)/8 h darkness (night).

Materials and Methods. To date, there is no reference data for application of dry sterilization of the start seeds in in vitro propagation of Gesneriaceae family. For the first time, this method has been applied to the Gesneriaceae family - the Ramonda and Haberlea genera, which is an original contribution of our research team. Sterilized seeds are put in vitro in vials with a nutrient medium GM (7). They germinate (Fig. 2A) and epycotyls of the 3 weeks old seedlings are transferred on RA medium for micropropagation. As a basic protocol for Ramonda serbica Pan. micropropagation, we use the one applied in (7) for Ramonda myconi (L.) Rchb.

Fig. 2. ) Seed germination after dry sterilization; B) Single microplant, grown from a seed; C) A buldge, containing multiple microplants.

Results and Discussion

During the micropropagation stage, the microplants grow on a medium with phytohormones (BAP and IAA) and an antioxidant added. The combination of BAP and IAA hormones activates the development of the accelerated buds and stimulates tissue differentiation. The antioxidant (glutathione) suppresses the tissue necrosis and accelerates the growth. As as a result, a buldge containing multiple microplants is formed (Fig. 3). The microplants from the buldge are then separated under sterile conditions in a laminar box. Thus, the microplants can be multiplied until the required number is reached (Fig.3 B-F). Part of the plants is stored as a live collection of in vitro plants, while other can be used as model plants for multidisciplinary research and education.

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Fig. 3. Micropropagation of Ramonda serbica Pan .

At the rooting stage the growing microplants are transferred on the same nutrient medium but only a phytohormone IAA from the auxin group is added. At the end of this stage, the microplants form a stabile root system. During adaptation, the rooted plants are planted in vivo in a turf-perlite mixture and they are grown under controlled air humidity and temperature. Our team has achieved successful adaptation of Ramonda serbica Pan. at the Plant Biotechnology laboratory of the University of Plovdiv (Fig.4).

biological diversity 2005-2010, we propose conservation of the Ramonda serbica Pan., habitat Montana district, Bulgaria as a live collection of in vitro plants at the Plant biotechnology laboratory of the University of Plovdiv. This species has not been yet conserved as a in vitro live collection in Bulgaria. For the first time, the dry sterilization method has been applied to the Gesneriaceae family - the Ramonda and Haberlea genera, which is an original contribution of our research team. The live collection of Ramonda serbica Pan. in vitro plants will contribute to the following directions: Conservation and sustained management of the biodiversity - reintroduction of this endangered species in its natural habitat. The live collection of Ramonda serbica Pan. in vitro plants will be used as year-round donor of model plants for multidisciplinary research and education purposes. Acknowledgements This research is supported by the grant DO 02/236 National Science Fund at the Ministry of Education and Science, Bulgaria.

Fig. 4. Ramonda serbica Pan. adapted plants

In accordance with the National plan for protection of

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