Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh, India.
Plant Cell Rep. 2010 May;29(5):495-502. doi: 10.1007/s00299-010-0838-4. Epub 2010 Mar 9.
Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies.
重组基因赋予抗生素或除草剂抗性，被广泛用作植物转化中的选择标记，以选择最初的转基因事件。然而，一旦转基因植物得到开发和鉴定，这些标记就变得多余了。尽管没有证据表明选择标记基因对消费者和环境不安全，但如果可以从最终的转基因事件中消除标记基因，那将是理想的。有效的转化方法的可用性可以使开发不含有标记基因的转基因事件成为可能。利用花生的高效转化潜力，我们报告了一种不使用任何选择标记基因开发其转基因的技术。构建了含有玉米（Zmpsy1）类胡萝卜素合酶基因或水稻（Rchit）几丁质酶基因的无标记二元载体，并用于根癌农杆菌介导的花生转化。在体外生长的假定转基因事件最初通过 PCR 进行鉴定，并通过点印迹分析、Southern 印迹和 RT-PCR 进一步确认基因整合和表达，它们显示出超过 75%的转化频率。该系统简单、高效、快速，不需要复杂的分离步骤和选择转基因事件的分析。这种生成无标记转基因植物的方法最大限度地降低了引入不需要的遗传变化的风险，允许多个基因的叠加，并可适用于具有高芽再生效率的其他植物物种。
