Proceedings Volume 2010 (format .pdf) - SimpBTH

from Rila Mountain National Park of Bulgaria. Plant material was kindly suppliedby Dr. L. Evstatieva, Institute of Botany, Bulgarian Academy of Sciences. "Fresh"and "old" seeds were used to study germination efficiency in in vitro conditions."Fresh" seeds were cultivated in vitro in a short period (few days to 2 weeks) afterharvesting. "Old" seeds were used after being stored at room temperature from 6 to12 months.Explants for in vitro propagation were obtained from twenty days old seedlingsby cutting the seedlings and obtaining apical buds (6-8 mm in size) and stemsegments (8-10 mm in size) with a leaf node and two adjacent leaves. Fifty to 100seeds or 80-120 explants were tested for each experimental variant. Statisticalanalysis was according to Sigma Plot 3.1. (Systat Software Inc – SSI, A scientificData Management Company).Decontamination. Seeds were subjected to decontamination by different time oftreatment with various sterilizing agents applied in single or in a consecutivemanner according to the following five schemes:(1) 96 o C 2 H 5 OH for 5 min;(2) 70 o C 2 H 5 OH for 3 min; 20 % [v/v] bleach (trade mark Ache produced byProcter & Gamble Co., USA containing 5 % of active chlorine) for 15 min;(3) 70 o C 2 H 5 OH for 1 min; bleach for 5 min;(4) 70 o C 2 H 5 OH for 1 min; bleach for 10 min;(5) 70 o C 2 H 5 OH for 1 min; 0.2 % HgCl 2 for 10 min.A drop of Twin-20 (Sigma-Aldrich) was added into the decontaminationsolution.Each procedure was followed by triple rinse in autoclaved distilled water for 5 min,10 min and 15 min.Media composition for in vitro cultivation. Seeds were germinated on filterpaper soaked in distilled water (control conditions) or on Murashige and Skoog(1962) basal medium (MS) supplemented with 10 - 100 mg/l gibberellic acid, 30g/l sucrose and 6 g/l agar-agar (Table 1). MS basal medium enriched with 2 mg/lzeatin, 0.2 mg/l IAA, 0.4 mg/l GA 3 , 30 g/l sucrose and 7 g/l agar-agar for shootdevelopment. Rooting media contained half strength MS salts, 10 g/l sucrose and 6g/l agar-agar and different phytoregulators (Table 1). The pH of all media wasadjusted to 5.7 - 5.8 prior to autoclaving at 1.1 kg cm -2 , 121 o C, for 20 min.Conditions for in vitro cultures and plant adaptation. In vitro materials werecultured in a phytothrone room at 16 h photoperiod, temperature of 18-21 o C and40 µMm -2 s -1 (fluorescent lamps type FL-40 W-1, Svetlina Ltd., Bg). Regeneratedplantlets after formation of roots in vitro were transferred to small pots containingdifferent substrates (soil, peat and perlite only or in combinations) and were grownin chambers under different conditions (humidity from 60 % to 90 %, temperaturefrom 18 o C to 21 o C, light intensity 60 µMm -2 s -1 ).105