Development of in vitro cultures from Astragalus thracicus Griseb and Astragalus aitosensis (Ivanisch.) and evaluation of their bioproduction potential.

Two endemic Astragalus species - A. thracicus and A. aitosensis were investigated to establish and develop their in vitro cultures. We managed to optimize the in vitro growth conditions, suitable for growing and maintaining stable and long-lasting callus cultures. Furthermore, we measured the behavior and flavonoid biosynthetic potential of callus cultures after stress induction with selected elicitors methyl jasmonate (MJ), salicylic acid (SA), and yeast extract (YE). In case of bioticstress, we concluded that growth index of callus cultures from A. thracicus decreased inversely proportional to the concentrations of elicitors applied, as SA showed the strongest growth inhibiting effect. The synthetic potential of the same callus cultures was evaluated based on HPLC analysis of levels of three flavonol aglycons – kaempferol, quercetin, and methylquercetin. The highest amount of kaempferol (9.12 µg/g dry weight) and quercetin (5.72 µg/g dry weight) was detected when callus cultures were exposed to YE at a concentration of 50 mg/l in medium for 72 hours. For the synthesis of the third aglycon studied, methylquercetin, the best inductive conditions (equivalent to 4.69 µg/g dry weight) were measured in media supplemented with MJ at a concentration of 200 µmol/l for 72 hours. The fastest kaempferol synthesis was found in the medium supplemented with 1000 µmol/l SA at 24 hours, quantified as 7.61 µg of kaempferol per gram of dry weight. Based on all these experiments, the study resulted in a development of a micropropagation protocol.