An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)
An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)
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DOI：
10.1007/s00299-010-0838-4
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发表时间：
2010-05-01
影响因子：
6.2
通讯作者：
Sharma, Kiran K.
中科院分区：
文献类型：
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作者：
Bhatnagar, Madhurima;Prasad, Kalyani;Sharma, Kiran K.
赋予抗生素或除草剂抗性的重组基因被广泛用作植物转化中的选择标记，用于选择初级转基因事件。然而，一旦转基因植物被开发和鉴定，这些就变得多余了。尽管没有证据表明选择性标记基因对消费者和环境是不安全的，但如果标记基因可以从最终的转基因事件中消除，则将是期望的。有效转化方法的可用性可以使开发前期缺乏标记基因的转基因事件成为可能。利用花生高而稳定的转化潜力，我们报道了一种不使用任何选择标记基因的转基因技术。构建了含有玉米八氢番茄红素合成酶基因（Zmpsy 1）和水稻几丁质酶基因（Rchit）的无标记双元载体，并用于根癌农杆菌介导的花生遗传转化。在体外生长的推定的转基因事件最初通过PCR鉴定，并通过斑点印迹分析、Southern印迹和RT-PCR进一步确认基因整合和表达，其中它们显示超过75%的转化频率。该系统简单、高效、快速，并且不需要复杂的分离步骤和用于选择转基因事件的分析。这种用于产生无标记转基因植物的方法使引入不需要的遗传变化的风险最小化，允许堆叠多个基因，并且可以适用于具有高芽再生效率的其他植物物种。
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.