**6. Transgenic** *AtCKX* **centaury plants under salinity stress** *in vitro*

Taking into account that centaury plants can inhabit saline soils in natural habitats and because it is known that CKs play an essential role in the salinity stress response, the effect of NaCl-induced stress on regeneration potential of centaury shoots and roots was evaluated. The investigated non-transformed as well as *AtCKX1* and *AtCKX2* transgenic lines showed different salinity tolerance to graded NaCl concentrations. In general, a higher salinity tolerance was found for roots compared to shoots. Furthermore, elevated NaCl concentrations in the culture medium had no inhibitory effect on centaury shoot growth. All centaury shoots, regenerated on media supplemented with different NaCl concentrations, showed similar morphology as shoots regenerated in solid root cultures grown on NaCl-free media. Similarly, Šiler et al. [134] demonstrated that centaury plants keep the rosette forms under salt stress *in vitro*. Variations in the salt tolerance could be explained by differences among centaury genotypes, which is related to the recent report describing this species as a common salt-tolerant medicinal herb from Mediterranean region [135]. Whereas the non-transformed and *AtCKX1* transgenic line showed the same trend of shoot regeneration potential under salt stress conditions, the *AtCKX2* transgenic lines differed exhibiting a gradually decreased frequency of regeneration, the average number of regenerated shoots and fresh shoot weight with increased NaCl in the medium. The *AtCKX* transgenic centaury lines differed in their salinity tolerance from the nontransformed control indicating possible involvement of CKs in this process.

Beside morphological traits, biochemical characterisation of *AtCKX* transgenic centaury plants was determined. Amount of endogenous proline is considered one of the factors involved in the plant stress tolerance. Accordingly, an increase in the proline content was found in all *AtCKX* centaury shoots and roots grown on graded NaCl concentrations. Evaluation of malondialdehyde (MDA) level and hydrogen peroxide (H2O2) in centaury plants grown *in vitro* during salt stress also provided evidence of their oxidative stress tolerance. In all centaury *AtCKX* shoots and roots, MDA and H2O2 contents were increased at graded NaCl concentrations. These findings correspond well with literature data demonstrating an enhancement of MDA and H2O2 contents under salt stress [136, 137].

On the other hand, *AtCKX* transgenic centaury plants showed altered reactive oxygen species (ROS) homeostasis. It is rather difficult to summarize and conclude how salt stress affected on antioxidative enzymes activity. An increase of the superoxide dismutase (SOD) activity was detected in all centaury plants grown on graded NaCl concentrations. Interestingly, the SOD activity was always higher in roots than in shoots. Enhanced catalase (CAT) and peroxidase (POX) activities were detected in centaury plants grown on graded NaCl concentrations as well.

Similarly to centaury, transgenic *AtCKX2* tobacco plants showed increased SOD and CAT activity in comparison to wild type while lower SOD activity was found in roots than in shoots under salt stress *ex vitro* [118]. Increased SOD, CAT and POX activities in salt-stressed non-transformed and *AtCKX* transgenic centaury plants indicates a crucial role of these antioxidant enzymes in protecting plant tissues from superoxide radical and hydrogen peroxide and suggests their association with improved tolerance of plants to environmental stress conditions.

Evidently, the salinity stress caused morphological, physiological and biochemical changes in both non-transformed and *AtCKX* transgenic centaury plants. Elevated proline, MDA and H2O2 contents as well as increased antioxidative enzymes activities confirmed that centaury plants are trying to overcome salt stress conditions to maintain or re-establish their normal growth and development.
