Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima
Genetic transformation of Centaury (Centaurium erythraea Rafn.) using AtCKX1 and AtCKX2 genes
Doctoral thesis (Published version)
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Plant species Centaurium erythraea Rafn., commonly known as common centaury, has been used for centuries to cure febrile conditions, regulate blood sugar, treat anemia, jaundice and gout and to increase appetite and stimulate digestion. Because of its numerous therapeutic properties centaurii herba is officially recognized as a drug in a number of pharmacopoeias. Root tips of ≈10 mm average length, isolated from seedlings obtained under sterile conditions, were used as initial explants in this work. The in vitro culture of non-transformed centaury roots was established on solid half-strength MS nutrient medium without growth regulators. Organogenesis is a spontaneous process in centaury root culture. The first changes were noticeable on the basal part of the initial explants, after seven days of growth on MS medium. The root explants thickened and began to burst over the entire length of the basal part. During four weeks of subculture, a large number of adventitious buds were observed on initial explants. In order to obtain transgenic centaury plants with lowered endogenous cytokinin levels, in vitro cultivated root explants were transformed with Agrobacterium tumefaciens GV3101 bearing a pBinHTX plasmid with either AtCKX1 or AtCKX2 genes encoding isoforms of cytokinin oxidase/dehydrogenase (CKX). PCR analysis of genomic DNA confirmed the effecient integration of both AtCKX1 (30%) and AtCKX2 (28.2%) genes. In order to investigate the expression of AtCKX genes in transgenic centaury plants, an optimization of PCR protocol with qPCR primers was necessary. Optimization of PCR protocol implicated finding the optimal annealing temperature and simultaneous application of different PCR additives and enhancers. Addition of 5% DMSO significantly improved the specificity of qPCR amplification of AtCKX1 and AtCKX2 genes. qRT-PCR analysis indicated that AtCKX1 and AtCKX2 genes were expressed in 50% and 64% of the analyzed transgenic lines, respectively. In this work we clearly demonstrate a significant effect of AtCKX genes on cytokinin metabolism in transgenic centaury plants. Since CKX is the only known enzyme involved in cytokinin catabolism, it is considered a key factor in controlling the cytokinin levels in plant tissues. CKX enzyme activity was determined in shoots and in roots of all AtCKX transgenic lines. The analysis of the content of total cytokinins and individual groups of endogenous cytokinins in non-transformed centaury plants enabled, for the first time, a determination of cytokinin profile of this species in vitro. In this work we also showed that changes in the cytokinin metabolism have unpredictable effects on the endogenous IAA levels in plant tissues. The expression of AtCKX genes also affected some morphological traits of the transgenic centaury plants. We observed that a decrease of endogenous cytokinins caused a reduction of morphogenetic potential of transgenic centaury plants but did not significantly affect the biomass production in comparison to control plants. Analysis of photosynthetic pigments of centaury indicated that a decrease of endogenous cytokinins might be a prerequisite rather than a signal for the onset of senescence. The main active pharmacological compounds of the centaury are secoiridoids and xanthones. The content of the secoiridoids found in plants grown in vitro was similar to that in plants collected from nature, whereas the quantity of xanthones was significantly elevated in plants grown in vitro, in comparison to plants from nature. Most of the obtained AtCKX transgenic centaury lines produced less secoiridoids than plants from natural habitat or plants grown in vitro. On the contrary, it was determined that the majority of the AtCKX transgenic centaury lines accumulated more xanthones than plants grown in nature. As xanthones are increasingly being used for their pharmacological properties, AtCKX transgenic centaury plants could be used as a useful source of plant material for the production of novel drugs.
Keywords:Centaurium erythraea Rafn.; Agrobacterium tumefaciens; qRT-PCR; Genetic transformation; AtCKX genes; CKX activity; Cytokinins; Secondary metabolites; Secoiridoids; Xanthones
Source:University of Belgrade, Faculty of Biology, 2012, 1-177
APA:Trifunović, M. (2012). Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima. University of Belgrade, Faculty of Biology. p. 177.
Vancouver:Trifunović M. Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima [#dissertation]. Belgrade: University of Belgrade, Faculty of Biology; 2012. 177 p.
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