New Researches in Biotechnology - Facultatea de Biotehnologii ...

Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011homeopathy and cosmetics. The plant is used as anti-inflammation drug stimulating woundhealing. It possesses also bacteriostatic, fungistatic, antirheumatic, cardiotonic andantihyperlipidemic effects (Willuhn, 1998). At present circumstances - law protected statusand enhanced pharmacy interest it is necessary to elaborate methods for preservation ofvaluable clones of this species.The objective of the present study was to develop an effective protocol for longtermin vitro conservation of A. montana under slow growth conditions allowing the storageof this endangered plant.2. MATERIALS AND METHODSProlonged in vitro conservation of plant material: The A. montana shoots were obtainedfrom in vitro seedlings of German population (Botanical garden, Chemnitz). The plantmaterial was maintained on rooting medium (basal medium containing half-strength MSnutrients salts supplemented with 0.5 mg/l IBA) in order to align the phase of growth anddevelopment. Retarding the growth of cloned plants was accomplished through:- Using the half-strength MS medium- Reducing the concentration of sucrose- Addition of osmoticum (mannitol)- Cultivation under low light intensity (20 µMm -2 s -1 )For the experiment, A. montana plants were cultured on ½ MS medium containing 2%sucrose, 0.5 mg/l IBA and different addition of mannitol (0; 1; 2; 3 and 4%). The mediumwithout mannitol was used as control. The plants were placed vertically in the glass tube(150 x 20 mm), containing 8 ml of rooting medium. One shoot per culture tubes wasinoculated and each treatment involved 20 plants.The treatments were repeated two times.A survival of plants was monitored each month within six months without subcultivation.The survival of prolonged storage in vitro cultures was determined by the presence of greenplants with healthy growing tips without necrosis. The percentages of rooted plants, heightof vegetative part of plants and root length were recorded during storage (1, 3 and 6 monthof culture).Multiplication of A. montana plants after long-term storage plant material: Therecovery of plants after prolonged cultivation on medium with mannitol was initiated byculturing under optimal conditions for growth and development. The plants weresubcultured on two different propagation media (full strength MS medium supplementedwith 1 mg/l BAP or combination of 1 mg/l BAP and 0.1 mg/l IAA). The mean number ofshoots per explant after 3 and 6 months of in vitro conservation was recorded. The datawere statistically analyzed using Sigma Stat computer package (Sigma Stat 3.1, SystatSoftware, San Jose, California, USA).Culture conditions: All media were adjusted to pH 5.6 before autoclaving. The nutrientmedia were sterilized and autoclaved at 120 ºC for 20 min at 1 atm. The cultures werestored at 22 ± 2 °C (16/8h photoperiod) and low light intensity (20 µMm -2 s -1 ) for in vitroconservation of plant material and light intensity (40 µMm -2 s -1 ) for recovery of plants afterprolonged cultivation.57