Transformation methods for obtaining marker-free genetically modified plants
Transformation methods for obtaining marker-free genetically modified plants
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DOI：
10.1002/9780470958988.ch15
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发表时间：
2011-03
期刊：
影响因子：
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通讯作者：
J. Schaart;F. Krens;A. Wolters;R. Visser
中科院分区：
文献类型：
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作者：
J. Schaart;F. Krens;A. Wolters;R. Visser
在世界范围内，对植物产品的需求不断增长，特别是食品和生物燃料。这种需求是由于世界人口的增加、消费模式的改变以及使用持久能源的政治意识造成的。为了满足日益增长的需求，增加农业生产是必不可少的。为了应对这一问题，迫切需要在各个层面（产量、可提取物质质量）对作物进行改良，并使作物适应更不利的条件（干旱、盐度、低氮有效性、不同温度）。这些改进原则上可以通过传统育种来实现，但这对许多作物来说是一个耗时的过程。作为一种相对快速的替代方法，特别是在无性繁殖或长世代异花授粉的植物中，可以使用转基因方法对作物进行改良。然而，有益基因（或等位基因）的可用性是成功开发这种基因改良作物的先决条件。因此，重要的是要认识到，大规模基因组测序项目带来的DNA序列信息数量的迅速增加，加上我们对基因功能的了解不断扩大，使得寻找有益（候选）基因的方法更加直接。此外，对于一些作物，已经制定了优化的转化方案（例如，见Curtis 2004），允许通过基因改造快速改善这些物种。这些转化方案大多采用可选择的标记基因，如抗生素抗性或除草剂抗性基因，用于转化细胞的选择性生长。然而，公众对转基因（GM）植物的环境和食品安全问题的关注（例如，见Halford和Shewry 2000）导致了对没有可选择标记的转基因植物生产技术的需求。在这里，我们描述了两种不同的方法，开发内部，获得无标记的转基因植物，而不需要遗传分离。这两种方法都特别适用于无性繁殖作物（如马铃薯和许多水果作物）或世代时间较长的作物（如木本植物）中生产无标记的转基因植物。第一种方法涉及不使用任何可选择标记基因的转化，而第二种方法采用位点特异性重组介导的基因切除，用于选择转基因植物或组织。
Worldwide, there is a growing demand for plant products, in particular, for food and biofuels. This demand is caused by an increasing world population, changes in consumption patterns, and the political awareness to use durable energy sources. An increase of agricultural production is essential to meet the growing demands. To confront this, there is an urgent need for crop improvement at various levels (yield, quality extractable matter) and adaptation of crops to more adverse conditions (drought, salinity, low nitrogen availability, different temperatures). These improvements can in principal be achieved by classical breeding, but this is for many crops a time-consuming process. As a relatively fast alternative, especially in vegetatively propagated or cross-pollinated plants with long-generation times, crops may be improved using a genetic modification approach. The availability of beneficial genes (or alleles), however, is a prerequisite for the successful development of such genetically improved crops. Therefore, it is important to realize that the rapidly increasing amount of DNA sequence information coming from large-scale genome sequencing projects, combined with our expanding knowledge of gene functions, has enabled a more direct approach to search for beneficial (candidate) genes. Besides, for several crops, optimized transformation protocols have been developed (e.g., see Curtis 2004), allowing a rapid improvement of these species through genetic modification. Most of these transformation protocols employ selectable marker genes, such as antibiotic resistance or herbicide resistance genes for selective growth of transformed cells. However, public concerns on the issue of the environmental and food safety of genetically modified (GM) plants (e.g., see Halford and Shewry 2000) have led to a demand for production technologies for GM plants without selectable markers. Here, we describe two different methods, developed in-house, for obtaining marker-free GM plants without the need for genetic segregation. Both methods are in particular suitable for production of marker-free GM plants in vegetatively propagated crops, such as potato and many fruit crops, or in crops with a long-generation time, such as woody species. The first method concerns transformation without the use of any selectable marker gene, whereas the second method employs site-specific recombination-mediated excision of the gene used for selection of transgenic plants or tissues.