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Lecturer(s)
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Sojka Jiří, Mgr. Ph.D.
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Šamajová Olga, Mgr. Dr.
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Luptovčiak Ivan, Mgr. Ph.D.
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Polláková Kateřina, Mgr.
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Buzáš Marek, Mgr.
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Course content
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Content of the seminar: 1) Definition of basic concepts, history of transformation and transgenosis of plants, bacteria Agrobacterium tumefaciens - Ti plasmid. Distribution of scientific papers to students for journal clubs. 2) The principle of transformation using A. tumefaciens: the transfer of T -DNA into the plant. Other species of the Agrobacterium genus. 3) Stable vs transient transformation of plants: transgenosis mono- and dicotyledonous plants, methods of transient transformation of leaves, cotyledons, protoplasts. Methods for stable transformation of plants. 4) Components of transgenes - promotors, terminators, polyadenylation signal, 5'not coding region. 5) The gene silencing - a T-DNA insertional mutagenesis, RNAi technique. 6 ) Reporter genes and marker lines. Selection markers for plant transgenes - antibiotics, herbicides, carbohydrate source and plant hormones. 7 ) Loss of activity of transgenes - the transcriptional and post-transcriptional inactivation of the transgene following Journal club I 9) GMO - tolerance to herbicides and insect pests, protease inhibitors. Journal Club II 10) GMO - other transgenes used nowadays. GMO field research. Journal club III 11) GMO legislation in our country. GMO vs Eko -Bio sustainable agriculture, final discussion Journal Club IV 12) Final test Content of the practical seminars: 1) Arabidopsis thaliana handling. Practical part: Arabidopsis thaliana sawing - Columbia ecotype for stable floral-dip transformation. 2) E. coli a A. tumefaciens cultures, content of the media Practical part: preparation of medias for A. tumefaciens, inoculation of A. tumefaciens to the liquid and solid media 3) Transient transformation - methods overview Practical part: transient transformation of N. bentamiana leaves with A. tumefaciens 4) Practical part: microscopical observation of transformed N. benthamiana leaves 5) Practical part: transient transformation of A. thaliana seedlings with A. tumefaciens 6) Practical part: microscopical observation of transformed A. thaliana seedlings 7) Methods for bacterial DNA isolation Practical part: isolation of bacterial DNA. 8) Stable "Floral-Dip" transformation of A. thaliana plants. Practical part: stable "Floral-Dip" transformation of A. thaliana plants (prepared during the first practical seminar). 9) Practical part: A. thaliana seeds handling: AraSYSTEM. 10) Selection markers for plant transgenes. Practical part: preparation of selection media for A. thaliana seeds - sterile handling and sowing of transformed seeds. 11) Practical part: selection of transformed plants 12) Final discussion and protocols corrections.
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Learning activities and teaching methods
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Monologic Lecture(Interpretation, Training), Dialogic Lecture (Discussion, Dialog, Brainstorming), Observation, Demonstration, Training in job and motor Skils, Laboratory Work
- Attendace
- 40 hours per semester
- Semestral Work
- 10 hours per semester
- Preparation for the Course Credit
- 20 hours per semester
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Learning outcomes
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The course aims to introduce students to the fundamental principles of plant genetic transformation and the applications of transgenic technologies in both basic and applied research. Students will acquire theoretical knowledge of methods for the transfer of genetic information into plant cells, the principles underlying the generation of transgenic plants, and their use in the study of gene function as well as in the targeted modification of agronomically important crop traits. The introductory part of the course covers the fundamentals of plant transgenesis, including the principles of transient and stable genetic transformation. Particular attention is devoted to plant cell transformation mediated by Agrobacterium tumefaciens and to direct methods of DNA delivery into plant cells. The subsequent part focuses on the application of genetic transformation in basic research. Students will become familiar with approaches for targeted modification of gene expression and functional gene analysis, including T-DNA insertional mutagenesis, RNA interference (RNAi), reporter genes, selectable markers, and other molecular biological techniques commonly used for gene function characterization. In the area of applied research, the course addresses the use of transgenic technologies in plant breeding, the development of genetically modified (GM) crops, strategies for improving resistance to biotic and abiotic stresses, emerging transgenic technologies, and their significance for sustainable agriculture. The course also introduces the fundamental principles of the legislative regulation of genetically modified organisms, risk assessment procedures, and the legal framework governing the use of GMOs in the Czech Republic and the European Union. The practical component of the course is designed to provide hands-on experience with fundamental plant transformation techniques. Students will become familiar with the stable transformation of the model plant Arabidopsis thaliana using the floral dip method and with the transient transformation of Nicotiana benthamiana by agroinfiltration. During laboratory exercises, students will learn how to prepare and cultivate Agrobacterium cultures, perform transformation procedures, and subsequently analyze the expression of fusion proteins using fluorescence microscopy. Practical training also includes the isolation of protoplasts from transformed N. benthamiana leaves, selection of transformed seeds and plants of A. thaliana, evaluation of transformation efficiency, and processing of microscopy-derived image data. During the seminar, students will present results obtained during laboratory exercises, interpret experimental data, and actively participate in scientific discussions.
After completing the course, the student should be able to understand the principles of genetic transformation of plants, independently master the most commonly used transient and stable transformation methods used in plant biotechnology practice, and be familiar with the basic methodological, application, and legislative aspects of plant biotechnology.
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Prerequisites
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unspecified
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Assessment methods and criteria
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Written exam, Final project
Attendance at all practical classes (100%), presentations of assigned publications and active participation in Journal club discussions, preparation of protocols from practical courses and successful completion of the credit test.
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Recommended literature
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PDF prezentace z jednotlivých seminářů.
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Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16. 1998.
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Davis AM, Hall A, Millar AJ, Darrah C, Davis SJ. Protocol: streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant methods, 5, 3. 2009.
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J.-F. Li, E. Park, A.G. von Arnim, A. Nebenführ . (2009). The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5.
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Jim M. Dunwell, Andy C. Wetten. (2012). Transgenic plants-methods and protocols. New York.
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Ondřej M., Drobník J. (2002). Transgenoze rostlin. Akademia.
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Wang K. (2006). Agrobacterium protocols, 2-nd edition. Humana Press.
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Wegel D. and Glazebrook J. (2002). The Arabidopsis Book. New York.
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