Plant Transformation and Transgenic Plants
1 The general aim of transformation, methods and a review of the past.
2. Introduction into the special tissue culture methods used during the process of transformation. Generating protoplasts. Plant regeneration from calli, and zygote. Discussing the problem occurs during tissue culture, like somaclonal variation and the methods to reduce the risk.
3. Classification of plant transformation technologies, introduction and a short review. Characterisation of Agrobacterium tumefaciens as a plant pathogen. Virulence genes, gene transfer, structure of the T-DNA, DNA integration into the plant genome.
4. Characterisation of different T-DNA vectors (binary and super binary) used for plant transformation. Assembly of the transformation vector and propagation in bacteria. Characterisation of all the steps, in details, carried out during agro bacterium mediated plant transformation.
5. Gene guns used successfully for plant transformation, so far, including the discussion of the advantages and disadvantages of the different equipment. Introduction into the method from DNA preparation till shooting. Parameters can be modified and needs optimisation for highly efficient callus transformation.
6. Chloroplast transformation. A brief introduction into the genome organisation of the chloroplast. Characterisation of the vectors, transformation cassettes used in this method. Gene transfer into the chloroplast genome step by step. How to solve problem may occur because of the code usage within the organelle?
7. Characterisation of the reporter genes used for plant transformation. Discussing the advantages and disadvantages of each reporter genes used so far. Characterisation of the selection marker genes used for plant transformation. Discussing the advantages and disadvantages of each marker genes used so far.
8. Introduction into the method of “clean gene”. Why is it important and how can be achieved? Environmental friendly selection marker genes. Marker elimination techniques (for example MAT) and characterisation of the enzymes and border sequences used during this method.
9. Characterisation of the constitutive promoters used for plant transformation. Characterisation of tissue specific and other special, like inducible promoters applied in plant transformation cassettes. Effect of the 3’ UTR sequence for gene expression in plant.
10. Pattern of transgene integration into the genome during biolistic and agro bacteria mediated plant transformation. Relation between gene integration and gene expression. The effect of the copy number on the transgene stability in the genome. Gene silencing as a major problem in plant transformation.
11. Fields of application of GM plants, how where and what for? Characterising the possible usage of different GM plants, from experimental through studying the proof of concept till biofermentors. How bright is the future of the GM plant?
12-14. Effect of the presence of GM plants to the industrial practise, to the environment and to the society. Is it the technology of the future? What is the role of the scientists involved in plant transformation, the politicians and the media to inform and educate the society? What type of legislations are required to make the use of GM plants safe? What sorts of regulations have already been implemented in the USA and the EU?
John Flex Jackson, Hans F. Linskens: Genetic Transformation of Plants, Springer Science & Business Media, 2013
Leandro Peña: Transgenic Plants: Methods and Protocols, Springer Science & Business Media, 2005