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Epigenetic effects such as gene silencing and variable expression are unintended consequences of plant transformation, a problem that is present in the transformation of all plant species. There is not yet a reliable way to prevent epigenetic silencing; however, the probability of epigenetic effects may be reduced by choosing an appropriate method of transgene introduction into a plant cell. Most methods used in plant biotechnology, such as direct gene transfer and particle bombardment, result in the introduction of multiple DNA molecules and, as a consequence, multi-copy multi-locus insertion patterns. These multiple insertions may lead to variations in transgene expression, epigenetic silencing being the most extreme. In contrast, Agrobacterium-mediated plant transformation procedures rarely cause such unintended effects. In this chapter, we present advantages and disadvantages of the Agrobacterium-mediated plant transformation method as well as protocols for transformation of Arabidopsis generative tissues and tobacco seedlings as the most classical techniques in these model plants, i.e., vacuum infiltration of explants and floral dip methods. Moreover, epigenetic effects of transgenes such as silencing related to the position and insertion effects as well as effects of the regeneration procedure causing somaclonal variation will be briefly discussed.
Key wordsAgrobacterium T-DNA tagging Vacuum infiltration Floral dip Arabidopsis Tobacco Transgene silencing Position effect Insertion effect Somaclonal variation OE lines KO lines
The author would like to thank Prof. Barbara Hohn and her co-workers for establishing the protocol for Agrobacterium-mediated transformation of tobacco seedlings, Dr. Igor Kovalchuk for his advice and encouragement, and Dr. Valentina Titova for language revision.
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