**2. Methodology**

All of the *in vitro* culture experiments were performed with plant material originated from *C. erythraea* Rafn. seeds, obtained from Jelitto Staudensamen GmbH, Schwarmstedt, Germany. The plant tissue culture methods were used to establish a solid centaury root culture further utilizable in the genetic transformation process. In all *in vitro* culture experiments, half-strength MS hormone-free medium (½MS) [103] solidified with 0.7% agar and supplemented with 3% sucrose and 100 mg l−1 *myo*-inositol was used. The *A. tumefaciens* strain GV3101 harbouring the pBinHTX plasmid with either the *AtCKX1* or *AtCKX2* gene under the control of the 35S promoter with Triple X enhancer [79] was applied for the transformation of *C. erythraea* roots. Usual molecular biology analyses, including PCR and qPCR reactions, were performed to confirm the insertion of *AtCKX* transgenes into centaury genome. The CKX activity was determined by *in vitro* assays based on the conversion of [2-3 H]iP (prepared by the Isotope Laboratory, Institute of Experimental Botany AS CR, Prague, Czech Republic) to [3 H] adenine. Detection and quantification of endogenous phytohormones were performed using HPLC/MS system with TSQ Quantum Ultra AM triple-quadrupole high-resolution mass spectrometer


#### **Table 3.**

*The list of all plant materials and methods used during the investigation of transgenic AtCKX centaury plants grown in vitro.*

#### **Figure 5.**

*Illustrative presentation of transgenic AtCKX centaury plants production in vitro and all subsequent investigations conducted to date.*

(Thermo Electron Corp., San Jose, CA, U.S.A.) operated in the positive-SRM mode. Secondary metabolites including secoiridoids (swertiamarin and gentiopicrin) and xanthones (eustomin and demethyleustomin) were analysed on an Agilent series 1100 HPLC instrument with DAD detector and a reverse phase Zorbax SB-C18 analytical column. The secondary metabolite profile of transgenic centaury plants was determined to as certain whether these plants might serve as potential producers of anti-cancer compounds. The effect of non-transformed and transgenic *AtCKX* centaury methanol extracts on antibacterial and antifungal activity on bacteria and microfungi was also investigated. The salinity tolerance of non-transformed and *AtCKX* transgenic centaury shoots and roots to graded NaCl concentrations were tested separately *in vitro*. All plant material and methods used in investigations are presented in **Table 3**. The illustrative presentation of transgenic *AtCKX* centaury plants production *in vitro* and all further applied analyses are shown in **Figure 5**.
