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ISOLATION OF TRANSGENIC PLANTS NICOTIANA TABACUM AND SILENE VULGARIS
Kováčová, Viera ; Doškař, Jiří (referee) ; Vyskot,, Boris (advisor)
This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.
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Agrobacterium tumefaciens application
Kováčová, Viera ; Burdychová, Radka (referee) ; Babák, Libor (advisor)
This project focus on biotechnologic applications of bacterium named Agrobacterium tumefaciens, which is capable to mediate genetic transformation not only for plants. Main points of project are divided between description of basic natures and cytology of A. tumafaciens following by overview of usage A. tumefaciens in methods of modifications of diffrent cells. Target of paper about biotechnologic use of A. tumefaciens is confrontation of effectivity of this method of modification with else, summary of positiv benefits of its using for future development of human population and introduction of fundamental transformation methods of modification most important crop plants. Final part of project is brief summation of basic statement from statistics and legislature of genetic modified crops.
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Health risk and profit of genetically modified golden rice
KUČEROVÁ, Kateřina
People in developing countries very often suffer from severe vitamin A deficiency due to an insufficient and unbalanced diet. This vitamin is necessary, among other things, for the production of the visual pigment rhodopsin. According to the World Health Organization, up to half a million children go blind each year because of this deficit. Its deficiency also weakens the immune system and thus significantly increases the risk of death from various infectious diseases. The best solution to this deficiency would be if the vitamin was contained directly in the only food that these people get - in husked rice. There is already a special, genetically modified rice, into which has been introduced by a complicated and extensive modification of its DNA the entire metabolic pathway, which ensures the production of betacarotene in this crop. In the theoretical part I deal with the golden rice project and the origin of this genetically modified crop by the method of transgenesis, using bacteria of the genus Agrobacterium tumefaciens. Plasmids of this bacterium are able to incorporate parts of their genetic information into the target organism. Thanks to restriction enzymes, we can insert genes selected by us into plasmids, which we then introduce into a specific plant. But are these products safe? Opinions on these crops are very diverse, but one thing interests everyone: how to safely identify a genetically engineered plant? In the laboratory, these experiments can best be mastered on model material. Therefore, in the experimental part of my bachelor thesis I deal with the transgenesis of the model plant Nicotiana tabacum using a selected strain of Agrobacterium tumefaciens. These bacterial strains were provided to me from the private collections of the Institute of Molecular Plant Biology in České Budějovice. The aim of my work in the laboratory was to master the practical methodology of preparation of genetically modified plant and subsequent verification of the presence of introduced genes in the examined samples of the model organism. Specifically, genes for antibiotic resistance. DNA isolation, PCR amplification and electrophoretic assays were used for this purpose. The signal gene used in agroinfection was verified too. Also, it was monitored how many copies of the transgene were integrated into the research material during agroinfection.
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Human infectious diseases and GMO
KOBLIHOVÁ, Tereza
This bachelor thesis is focused on the detection of the transgenes in DNA of the genetically modified plants, especially on the detection of the transgene nptII. Moreover, it will discuss the determinations of the fission ratio (the amount of copies) of the injected transgenes into the next generation. The theoretical part of this bachelor thesis includes information about bacteria because some of their kinds are the reasons of human infection diseases, however, couple of them are used in genetic engineering for plant transformation at the same time. The diseases which are caused by bacteria are cured by antibiotics. Antibiotics are antimicrobic substances which are used to stop the growth of or exterminate these microorganisms. The theoretical part is especially focused on findings related to antibiotics and gene manipulated organisms (GMO). They are related to the concerns about using the selective gene nptII in the construct of these organisms, because this gene codes enzyme neomycin phosphotransferase II, which deactivates the effects of the Antibiotic called kanamycin. Organisms, where this gene is included are getting resistant to these antibiotics. The aim of the methodical part was the cultivation of own genetic modified plants carrying artificially injected genes for the resistance to kanamycin (nptII) and the signal gene GUS. Furthermore, we were looking for the subsequent determination of presence of the gene ntpII in the samples from these plants. The first necessary step to the determination of the gene nptII in the transgenic plants was multiplying of the DNA section containing this gene. That was accomplished by Polymerase Chain Reaction (PCR). After PCR was adapted, the detection of nptII gene in samples was made by gel electrophoresis. A histochemical test was used for the detection of the presence of the gene nptII in the plant samples and the confirmation of the transformation. As a model organism, the Nicotiana tabacum was chosen, which was transformed by Agrobacterium tumefaciens.
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Transformation of potato with protease inhibitor gene SPI-2
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Vítámvás, Pavel (referee)
The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....
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The role of atmospheric precipitation in anther dehiscence
Kampová, Anna ; Vosolsobě, Stanislav (advisor) ; Valuchová, Soňa (referee)
Anther dehiscence is an important process taking place at the end of the plant life cycle. This process consists of various follow-up steps which result in anther opening and pollen grains exposure. Good timing of the anther dehiscence must be synchronized with pollen grains maturation and flower opening. Atmospheric precipitation is a high-risk factor for the anther dehiscence. Male fitness of plants can be reduced when anthers open during poor weather conditions. The aim of this study was to investigate the effect of atmospheric precipitation, rain and dew, on Arabidopsis arenosa anther dehiscence. We observed that rain and dew led to a postponed final stage of the anther dehiscence. This caused delayed pollen release. The effect of aqueous and nonaqueous environment on the anther dehiscence was also tested. Experiments with transformation of A. arenosa using Agrobacterium tumefaciens were performed. Key words: anther dehiscence, flower opening, rain, dew, Arabidopsis arenosa, Agrobacterium tumefaciens, transformation
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Genetically modified plants in practical applications
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Cvrčková, Fatima (referee)
Genetic engineering (GI) of plants is a very current topic, and more and more controversial, since it is becoming an inseparable part of our lives. GI has, among other things, a great potential to help solve the current problem of hunger and malnutrition in certain parts of the world. The goal of this project is to clarify what genetically modified (GM) plants are, to present the possibilities of their practical use, to explain methods of preparation and to consider their advantages and eventual risks. By GM plant we understand a plant whose genetic information has been changed by introducing or removing part of the genetic information (a section of DNA, generally a gene). The most frequently used methods of transformation are the transformation by Agrobacterium tumefaciens and the balistic method. GM plants can be used for production of food or feed in better quality and greater quantity, thanks to an introduction of higher resistance, whether it is to biotic stress (pest, virus or bacterial resistance) or to abiotic stress (drought, salinity, toxic substance in the ground..). In particular, the resistance to abiotic stress is an important issue these days, since through the climate changes some regions suffer more and more from insufficient precipitation and consequent drought. Some of the GM plants grown...
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The role of atmospheric precipitation in anther dehiscence
Kampová, Anna ; Vosolsobě, Stanislav (advisor) ; Valuchová, Soňa (referee)
Anther dehiscence is an important process taking place at the end of the plant life cycle. This process consists of various follow-up steps which result in anther opening and pollen grains exposure. Good timing of the anther dehiscence must be synchronized with pollen grains maturation and flower opening. Atmospheric precipitation is a high-risk factor for the anther dehiscence. Male fitness of plants can be reduced when anthers open during poor weather conditions. The aim of this study was to investigate the effect of atmospheric precipitation, rain and dew, on Arabidopsis arenosa anther dehiscence. We observed that rain and dew led to a postponed final stage of the anther dehiscence. This caused delayed pollen release. The effect of aqueous and nonaqueous environment on the anther dehiscence was also tested. Experiments with transformation of A. arenosa using Agrobacterium tumefaciens were performed. Key words: anther dehiscence, flower opening, rain, dew, Arabidopsis arenosa, Agrobacterium tumefaciens, transformation
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Possibilities of synthesis of human glycoproteins in legumes seeds
Ploužková, Tereza ; Ryšlavá, Helena (advisor) ; Vaněk, Ondřej (referee)
The expression of recombinant proteins in seeds is especially advantageous due to the high concentration of storage proteins in a small volume. Recombinant proteins accumulate in seeds at high levels, remain stable for many years, and because of the low content of alkaloids and other substances in seed environment they can be easily extracted. There is also a minimal risk of contamination. This bachelor thesis deals with the testing of transient expression of gene constructs directly in maturing soybean seeds. In this work, we have have prepared gene constructs for strong transient expression of the uidA gene in plant tissues. The impact of the acetosyringone and dithiothreitol additives and the impact of their combinations on the transient expression and activity of the uidA gene have been studied as well. Furthermore, the optimal length and environment of sonication have been studied. Finally, the effectiveness of transient expression by in vitro and in vivo has also been compared. It has been confirmed that acetosyringone has a positive effect only on seeds that had not been previously sonicated. With prolonged sonication, the relative activity of the uidA gene decreases. Furthermore, the positive effect of dithiothreitol on seeds that had not been sonicated has been found. Higher values of the...
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