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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 20113. RESULTS AND DISCUSSIONProlonged in vitro conservation: The literature survey revealed that up to now theprotocol for in vitro conservation of A. montana under slow growth conditions have notbeen developed. The main requirement for prolonged in vitro storage is an effective methodfor micropropagation, as described previously (Petrova et. al. 2005, 2011). Table 1 presentsboth optimal propagation medium and rooting medium for in vitro conservation of A.montana plants.Table 1. Murashige and Skoog nutrient media modified for A. montana in vitro conservationNutrient mediumNutrient media for in vitro conservationcompositionMSP (for microMSR (for rooting)propagation)MS Full strength Half strengthSucrose (%) 3 2Agar (%) 0.6 0.6BAP (mg/l) 1.0IAA (mg/l) 0.1IBA (mg/l) 0.5Mannitol (%) 2The clonal propagation of plants can be saved valuable genotype, but the process neededplant subcultivation on fresh medium every months. In vitro conservation through retardingthe growth allows subcultivation at significantly longer periods (several months to year),which is more economically profitable (Negash et al. 2001). In our study slow growth ofplants was achieved by maintaining in vitro cultures in the presence of osmoticum(mannitol) at concentration 0; 1; 2; 3 and 4% and low light intensity (Table 2, Fig.1).Table 2. Effect of different concentration of mannitol on survival rate of A. montana plantsConcentrationof mannitol at½ МS nutrientmedium (%)After 1 month After 3 months After 6 monthsSurvival,%Rooting,%Survival,%Rooting,%Survival,%Rooting,%0 100 1001 90 100 90 952 65 80 65 70 65 553 55 40 40 354 20 058
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011a b c d eFig.1 A. montana plants maintened on ½ MS nutrient medium with different concentration of mannitol:а) Control; b) 1%; c) 2%; d) 3%; e) 4%The plants cultivated on ½ MS rooting medium (control) formed well developed rootsystem. The percentage of rooted plants reached 100% on the first month after inoculation.The plants needed a transfer to fresh medium because of their rapid growth (2.8 cm). Whenadding 1% mannitol to the medium the plants were viable and the percentage of survivaland rooting was 90 and 100% respectively, after one month of cultivation (Table 2). Thepresence of 1% mannitol initiated formation of 2-3 adventives shoots at the base of plant.On the other hand this concentration of mannitol caused growth reduction of vegetative partof plants (average height 1.7 cm), but the length of roots remained almost the same ascontrol (2.4 cm), allowing plants to be maintained for three months (Fig. 2). After thisperiod of time subcultivation on fresh medium was required. The addition of 2% mannitolestablished condition of osmotic stress, which reduced the growth rate and produced shortplants. Thus increased the time of subcultivation, but decreased values of all parametersinvestigated. This concentration of mannitol was retarded development of plants comparedto control. The survival rate decreased to 65% in the first month and remained constantuntil the end of cultivation. The stems were shortened, the leaves were with small size andthe development of root system was detained. This allowed subcultivations of plants aftersix months. The average height of plants was reduced to 1.1 cm. The differences betweenthe heights of control plants and those cultivated on MS medium supplemented with 2%mannitol were well proven statistically (Р≥0.01). The number of formed roots and theirlength were also reduced. The plants that didn’t form roots required an earlier transfer tofresh medium. The combination of 2% sucrose and 2% mannitol in ½ MS mediumprovided slow plant growth and increased the storage period of six months. It was foundthat this combination is optimal for in vitro conservation of plants maintained under lowlight intensity. The further increasing of mannitol concentration led to yellowing of leaves59
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