**5. Secondary metabolites in transgenic** *AtCKX* **centaury plants**

It is known that *in vitro* cultivation, although under controlled temperature and light conditions, stimulates increased secondary metabolites accumulation [124]. The previous investigations also showed that genetic transformation of centaury roots using *A. rhizogenes* affected the production of secondary metabolites [102, 125]. Considering that centaury plants were transformed using *A. tumefaciens* carrying *AtCKX* genes for the first time, it was interesting to reveal the effects of altered levels of endogenous CKs on secondary metabolites production of these transgenic centaury plants. The results of our analyses indicated modifications of the secondary metabolites production in this valuable medicinal plant species as a consequence of the *AtCKX* transgenes overexpression [105]. In methanol extracts of *AtCKX* transgenic centaury plants, the presence of bitter secoiridoids (swertiamarin and gentiopicrin) and xanthones (eustomin and demethyleustomin) was detected. The fact that *AtCKX* transgenic centaury plants produced the same compounds as non-transformed ones confirmed that there were no qualitative differences in specific secondary metabolites resulting from the *AtCKX* overexpression. On the other hand, quantitative changes in the secondary metabolite contents were found. Considering that gentiopicrin originates from swertiamarin in the metabolic pathway of iridoids, it is possible that increased activity of the swertiamarin → gentiopicrin converting enzyme stimulated gentiopicrin production. In all *AtCKX* transgenic centaury shoots, secoiridoid swertiamarin represented the predominant component. Still, in almost all centaury *AtCKX* shoots the content of swertiamarin was lowered in comparison to control shoots from *in vitro* cultures as well as from natural habitat. On the other hand, the production of the second bitter secoiridoid, gentiopicrin, was increased in the majority of *AtCKX* transgenic centaury shoots compared to control ones, grown *in vitro* and collected in natural habitat.

Unlike shoots, the *AtCKX* transgenic centaury roots produced decreased content of both secoiridoids, swertiamarin and gentiopicrin, in comparison to control roots grown *in vitro* and from natural habitat. It was shown previously that swertiamarin dominated in centaury shoots and roots from natural habitat while gentiopicrin prevailed in centaury shoots and roots grown *in vitro* [126–129].

Beside secoiridoids, the differences in xanthone content were also detected in *AtCKX* transgenic centaury plants. Most of *AtCKX* transgenic shoots and roots produced more eustomin and demethyleustomin than shoots from natural habitat but less than control *in vitro* shoots. The shoots and roots of only one transgenic centaury line, *AtCKX1*–29, produced significantly increased amount of both xanthones in comparison to both controls, shoots grown *in vitro* and those collected from natural habitat. It is important to note that shoots and roots of this transgenic line were characterised by a reduced level of bioactive forms of CKs [104].

### **5.1 Transgenic** *AtCKX* **centaury plants as potential producers of anti-cancer compounds and antimicrobials**

The methanol extracts of *in vitro* cultured *AtCKX1*–29 transgenic centaury plants were also tested for their antimicrobial effects [106]. Four Gram-positive, four Gram-negative bacteria and eight species of microfungi were selected for these *in vitro* investigations. The methanol extracts of non-transformed centaury plants, as well as pure secoiridoids (swertiamarin and gentiopicrin) and xanthones (eustomin and demethyleustomin), were used as a control. In general, the extracts of all tested centaury shoots and roots showed an adequate antibacterial activity on all tested bacteria. Methanol extracts of non-transformed and *AtCKX1*–29 transgenic centaury roots and pure gentiopicrin had a low antibacterial activity on *Micrococcus flavus, Escherichia coli* and *Enterobacter cloacae. Staphylococcus aureus* was found the most sensitive bacterial species in the *in vitro* assays. It was also shown that the extract of non-transformed centaury shoots was more effective on *S. aureus* than antibiotics, streptomycin and ampicillin. Interestingly, the pure compounds such as swertiamarin, gentiopicrin, eustomin and demethyleustomin exhibited higher antibacterial activity on all tested bacteria than centaury methanol extracts and commercial antibiotics (streptomycin and ampicillin) used as a positive control. High antimicrobial activity could be ascribed to bitter secoiridoid glycosides, which corresponds with previous literature data [130, 131]. Beside antibacterial impact, all tested methanol extracts of centaury shoots and roots also showed antifungal effects. Most of the applied compounds proved high antifungal activities. All pure secoiridoids and xanthones were more effective than methanol extracts against all fungi. In general, xanthone eustomin showed even 100 times higher antifungal potential than both of the applied mycotics (ketoconazole and bifonazole) used as a positive control*.* These results are significant as they represent the first report describing centaury xanthones as potential antimicrobials. In general, methanol extracts of non-transformed and transgenic *AtCKX1* centaury shoots and roots showed better antibacterial activity, while pure secoiridoids and xanthones were more active against fungi.

Antioxidant properties of centaury plants and pure xanthones were reported almost twenty years ago [132, 133]. Considering that transgenic *AtCKX1*–29 roots produced increased amounts of xanthones, their potential antioxidant activity was evaluated [106]. The transgenic *AtCKX1* centaury shoots and roots were found to exert higher antioxidant activity compared to non-transformed plants. The root extracts were two to five times more effective than those from the shoots. Transgenic *AtCKX1*–29 roots, containing the highest amounts of total phenolics, were the most effective the scavenging of the DPPH radicals. The antioxidant effects of transgenic centaury methanol extracts could be assigned to elevated levels of xanthone compounds. Accordingly, centaury plants with increased content of secondary metabolites could be of practical importance in developing novel drugs with a potential use in agronomy, veterinary, medicine and food industry.
