Structural, physiological and genetic diversification of Silene vulgaris ecotypes from heavy metal-contaminated areas and their synchronous in vitro cultivation.

Results provide significant comparison of leaf anatomy, pigment content, antioxidant response and phenolic profile between individuals from miscellaneous populations and describe unified cultivation protocols for further research on stress biology. The plant communities growing on heavy metal-polluted areas have attracted considerable attention due to their unique ability to tolerate enormous amounts of toxic ions. Three ecotypes of Silene vulgaris representing calamine (CAL), serpentine (SER) and non-metallicolous (NM) populations were evaluated to reveal specific adaptation traits to harsh environment. CAL leaves presented a distinct anatomical pattern compared to leaves of SER and NM plants, pointing to their xeromorphic adaptation. These differences were accompanied by divergent accumulation and composition of photosynthetic pigments as well as antioxidant enzyme activity. In CAL ecotype, the mechanism of reactive oxygen species scavenging is based on the joint action of superoxide dismutase and catalase, but in SER ecotype on superoxide dismutase and guaiacol-type peroxidase. On the contrary, the concentration of phenylpropanoids and flavonols in the ecotypes was unchanged, implying the existence of similar pathways of their synthesis/degradation functioning in CAL and SER populations. The tested specimens showed genetic variation (atpA/MspI marker). Based on diversification of S. vulgaris populations, we focused on the elaboration of similar in vitro conditions for synchronous cultivation of various ecotypes. The most balanced shoot culture growth was obtained on MS medium containing 0.1 mg l-1 NAA and 0.25 mg l-1 BA, while the most abundant callogenesis was observed on MS medium enriched with 0.5 mg l-1 NAA and 5.0 mg l-1 BA. For the first time, unified in vitro protocols were described for metallophytes providing the opportunity to conduct basic and applied research on stress biology and tolerance mechanisms under freely controlled conditions.