2.*Transfer of T-DNA into a plant cell*

VirD and VirE proteins play a significant role in the formation of single-stranded T-DNA, its cutting and transfer to the plant cell. The products of the VirD region (VirD1 and VirD2 proteins) are part of an endonuclease complex catalyzing specific single-stranded breaks that release a single-stranded T-DNA segment from the Ti plasmid. After excision, the T-chain shifts polarly and covalently binds the VirD2 protein. This protein protects the T-chain from destruction and remains there during transport to the plant cell. The transport of VirD2/T-DNK complex (immature T-complex) into the cytoplasm of the host cell, together with several other vir proteins (VirE2, VirF and VirE3), is performed through a specialized

membrane transport system. In cytoplasm of the plant cell, a mature T-complex is formed, which is protected from the action of nucleases during transport to the nucleus [15].

### 3.*T-DNA gene incorporation and expression*

The last, and probably the most critical step of genetic transformation, is the integration of T-DNA into the host genome. Integration into the plant genome occurs by non-homologous recombination in any part of the genome, taking preferentially place in regions with a higher percentage of adenine-thymine (AT) bonds [16]. Transgene expression depends on numerous factors such as e.g. the number of copies of an introduced gene and the site of transgene integration [17–19]. Stably integrated T-DNA is transferred to the next generation [20].

The mechanism of genetic transformation today involves the use of so-called "disarmed" *A. tumefaciens* strains, in which oncogenes are removed from the plasmids. The system of binary vectors usually used in genetic transformation processes divides the original Ti-plasmid into two plasmid vectors: an artificial (recombinant) Ti-plasmid carrying the gene of interest and an auxiliary plasmid [21]. In each binary plasmid, additionally to a foreign gene of interest the marker genes are also inserted and divided into selective and reporter genes [22]. The selective marker genes are most often prokaryotic genes that encode resistance to some antibiotics (kanamycin, hygromycin etc.). This negative selection implies using a selective medium which allows the distinction of transformed plant cells from non-transformed ones. Reporter genes are common but not necessary parts of the binary vector. These genes encode products which can be involved in the monitoring of transformation process (growth of transformed cells, regeneration efficiency and growth of transgenic plants). Visual reporter genes have been increasingly used because they do not require special artificial substrates for detection, and their expression is easily visually detected in tissue without destroying.

#### **1.2 Cytokinins**

Cytokinins (CKs) are plant hormones that individually or in combination with other hormones regulate numerous developmental and physiological processes in plants [23]. The name cytokinins comes from their originally discovered function, which is the stimulation of cell division – cytokinesis [24]. Cytokinins are divided into two major groups: natural and synthetic.

Natural CKs by chemical structure represent adenine derivatives. Depending on the component that binds to the adenine ring, natural CKs are further categorized into two groups:


*Transgenic* AtCKX *Centaury Plants Grown* In Vitro *DOI: http://dx.doi.org/10.5772/intechopen.94836*

#### **Figure 1.**

*Chemical structures of natural isoprenoid cytokinins.*

[26]. Substitution of the aromatic ring with a hydroxy group produces hydroxy derivatives such as *ortho*-topolin (oT) and *meta*-topolin (mT), while substitution with a methoxy group creates methoxy derivatives such as *ortho*-methoxytopolin (meoT) and *meta*-methoxytopolin (memT). Hydroxylation of the aromatic ring in *meta* position is a prerequisite for high CK activity [27, 28] and *meta*-topolin shows the most considerable effects in plants of all derivatives. The chemical structures of natural aromatic cytokinins are shown in **Figure 2**. Despite the isolation of the first naturally occurring aromatic CK as early as in the early 1970s [29], the distribution and function of aromatic CKs in the plant kingdom remain still unclear [30].

Synthetic CKs by chemical structure represent phenylurea derivatives. These substances show CK effects, however, to date they have not been discovered to be synthesized in plant tissues by normal metabolic pathways. The first identified synthetic CK was diphenylurea (DFU), discovered in 1955 [31]. The unexpected discovery of this compound stimulated the synthesis of numerous analogues such as CPPU (N-phenyl-N′- (2-chloro-4-pyridyl) urea) and TDZ (thidiazuron). These synthetic substances, presented in **Figure 3**, are highly stable and have stronger CK activity compared to zeatin [32–35].

#### *1.2.1 Cytokinin catabolism*

During plant development the levels of endogenous CKs are tightly metabolically regulated in plant tissues. The decreased amount of CKs is directly related to the increased activity of the catabolic enzyme, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12). This enzyme irreversibly degrades CKs by removing the *N*<sup>6</sup>  substituted side-chain to form adenine and the unsaturated aldehyde 3-methyl-2-butenal [36] (**Figure 4**). To date, CKX is the only known enzyme involved in the catabolism of specific CKs, and thus it represents a crucial factor in the control of CK levels in plant tissues. It is known that especially iP, *t*Z and their ribosides are sensitive to the CKX action. On the other hand, *c*Z is less sensitive [37] even

#### **Figure 2.**

*Chemical structures of natural aromatic cytokinins.*

**Figure 3.**

*Chemical structures of synthetic cytokinins.*

**Figure 4.**

*Chemical reaction of irreversible cytokine degradation by CKX enzyme.*

though some CKX isoforms exhibit high affinity for the *c*Z isomer as well [38]. CK *O*-glucosides, DHZ, aromatic CKs and their derivatives are believed to be the non-substrates for CKX [39–41]. By contrast, *Arabidopsis thaliana*, barley and maize CKX isoforms showed ability to degrade CK *N*9-glucosides [37, 42, 43].

According to the chemical structure, CKX enzyme is a flavoprotein with the flavin adenosine dinucleotide (FAD) bound domain [44, 45]. The activity of CKX in plant tissues is induced by exogenously applied CKs, both CKX substrates and non-substrates [46–48]. The CKX activity was first discovered in tobacco tissue [49], and subsequently this enzyme has been shown widely distributed in numerous plant tissues such as e.g. maize [50], *Vinca rosea* tumor [36], *Phaseolus vulgaris* callus [51, 52], wheat [53], and tobacco and poplar calli [47, 54]. The CKX enzyme is encoded by a small family of genes whose number varies from species to species. In *Arabidopsis thaliana*, seven genes responsible for the synthesis of CKX enzyme (*AtCKX1–7*) have been identified. These genes are expressed variously in different plant tissues [55, 56]. Individual *AtCKX* genes are expressed in the same tissues where CKs are synthesized (axillary buds, trichomes and vascular tissues) or in the nearby of these tissues. Genes encoding the CKX enzyme have been cloned also from numerous plant species (a detailed literature review is presented in **Table 1**).

#### **1.3 Genetic transformation of plants using** *AtCKX* **genes**

Genetic transformations of plants with specific genes encoding the CKX enzyme are beneficial for investigations of CK homeostasis. Thus, plants exhibiting increased overexpression of the *CKX* genes and a reduced amount of endogenous CKs can be obtained. Genetically engineered CKX plants can serve as a sound and valuable model system for studying physiological and morphological processes that are under control of CKs. To date, the impact of overexpression of different *CKX* genes on CK metabolism has been investigated in several plant species, as summarized in **Table 2**. The overexpression of *CKX* genes in tobacco and *A. thaliana* plants

#### *Transgenic* AtCKX *Centaury Plants Grown* In Vitro *DOI: http://dx.doi.org/10.5772/intechopen.94836*


#### **Table 1.**

*The list of CKX genes cloned and identified in different plant species.*


## **Table 2.**

*The list of plant species genetically transformed using CKX genes isolated from Arabidopsis thaliana.*

was demonstrated to affect significantly the phenotype of transformed plants, causing the *cytokinin deficiency syndrome*. In transgenic plants with this syndrome, apical dominance was decreased, shoots grew slowly, leaf size was reduced, the plants bloomed later and had a smaller number of flowers. Increased root growth size and activity of the root apical meristem (RAM) were observed. At the same time, decreased size and the shoot apical meristem (SAM) activity were also found. These symptoms were more evident in plants overexpressing *AtCKX1* transgene than in those with *AtCKX2* gene [55, 79]. As most of existing knowledge on regulation of CK levels and effects in plants have been obtained from studies of consequences of exogenously applied CKs, the specific CK-deficient transformants are expected to be more informative and to help in elucidation of the processes controlled by this group of phytohormones.

In plant shoots, CKs stimulate function of apical meristem and cell divisions. The stimulatory role of CKs on cytokinesis was discovered a long time ago [85]. Recently, it has been confirmed by analyses of transgenic *AtCKX A. thaliana* plants with reduced endogenous CK levels. Ultrastructural analysis of SAM cells showed clear cytological changes indicating detention of cell division and accelerated cell differentiation in transgenic CK-deficient *AtCKX A. thaliana* plants [86]. The development of vascular elements is also regulated by CKs. The optimal concentration of CKs in combination with auxins affects the formation of phloem [87]. In the shoots of transgenic *AtCKX A. thaliana* plants, xylem and phloem were reduced [55]. In addition, CKs control leaf formation and growth. Overexpression of the *CKX* transgenes in *AtCKX A. thaliana* plants significantly reduced leaf surface, which is undoubtedly related to cell divisions as previously described [55]. It has been shown that CKs are not able to completely prevent, but can significantly delay the senescence process [88]. Thus, it can be assumed that a reduced amount of endogenous CKs in CK-deficient plants accelerates senescence. Despite these expectations, the leaves of transgenic *AtCKX A. thaliana* plants did not show accelerated senescence [55]. The role of CKs during the plant reproductive development was approved by transgenic *AtCKX A. thaliana* plants, which formed a smaller number of flowers [55]. Enlarged embryos have also been observed in these plants, indicating a role of CKs in cell division during embryogenesis. The enlargement of embryos could be explained on the basis of *CKX* genes overexpression, occurring only in the later stages of embryogenesis [89].

Unlike the shoots, CKs inhibit the function of the RAM. Using the transgenic *A. thaliana AtCKX* plants, it has been confirmed that reduced CK levels strengthen the root system. The primary root growth is directly related to intense cell divisions in the root meristem of transgenic *A. thaliana AtCKX* plants [55]. The results obtained in these transformants are consistent with those by [90] demonstrating that exogenously applied CKs inhibited root elongation of untransformed plants by reducing the RAM size as well as reducing cell divisions. One would expect that the plants transformed with *CKX* genes have inhibited root branching. However, CK-deficient transgenic *AtCKX A. thaliana* plants surprisingly developed numerous lateral roots that elongated rapidly [55].

#### **1.4** *Centaurium erythraea* **Rafn.**

Common centaury, *Centaurium erythraea* Rafn. (syn. *C. umbellatum* Gillib and *C. minus* Moench), is the most known species from Gentianaceae family. Thanks to numerous pharmacological activities, centaury is listed as a medicinal plant species. In traditional medicine, centaury is used to treat febrile conditions, anaemia, jaundice, gout and to regulate blood sugar [91]. Bitter centaury juices have also been used for an increment of appetite, digestion stimulation and treatment of gastrointestinal tract diseases [92].

Nowadays, *C. erythraea* is rapidly disappearing from natural habitat and it is marked as an endangered plant species. Fortunately, even almost twenty years ago centaury showed vigorous regenerative potential *in vitro* [93–97]. The most interesting and essential information is that spontaneous morphogenesis of centaury is possible on nutrition medium without addition of any plant growth regulator [98]. In addition, during the years, centaury has also been used for studies of numerous developmental processes [99–101]. On the other hand, only few literature data exist describing genetic transformation of centaury. To date, most of Gentianaceae species, including *C. erythraea*, were usually genetically transformed only with *A. rhizogenes* [93, 102]. These investigations have mostly based and described the efficiency of plant regeneration and selection of transformed plant tissues. All of these previous investigations also encouraged us to use *A. tumefaciens* in genetic

transformation of centaury for the first time [84]. The successful production of transgenic *AtCKX* centaury plants allowed to use them as a suitable model for studies of numerous physiological and developmental processes under endogenous phytohormonal control.
