Ziemienowicz Alicja
Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada.
Methods Mol Biol. 2010;631:253-68. doi: 10.1007/978-1-60761-646-7_19.
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.
诸如基因沉默和可变表达等表观遗传效应是植物转化的意外后果，这一问题存在于所有植物物种的转化过程中。目前尚无可靠方法来防止表观遗传沉默；然而，通过选择合适的转基因导入植物细胞的方法，表观遗传效应的可能性或许可以降低。植物生物技术中使用的大多数方法，如直接基因转移和粒子轰击，都会导致多个DNA分子的导入，结果会形成多拷贝多位点插入模式。这些多个插入可能导致转基因表达的变化，其中表观遗传沉默是最极端的情况。相比之下，农杆菌介导的植物转化程序很少会引起此类意外效应。在本章中，我们将介绍农杆菌介导的植物转化方法的优缺点，以及作为这些模式植物中最经典技术的拟南芥生殖组织和烟草幼苗转化方案，即外植体真空渗透法和花浸染法。此外，还将简要讨论转基因的表观遗传效应，如与位置和插入效应相关的沉默以及导致体细胞克隆变异的再生过程的影响。
