Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics
Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics
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DOI：
10.1007/978-1-0716-0356-7_12
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发表时间：
2020-01-01
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影响因子：
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通讯作者：
Doherty, Angela
中科院分区：
文献类型：
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作者：
Sparks, Caroline A.;Doherty, Angela
以下方案描述了使用 BioRad PDS/1000-He 颗粒输送系统进行小麦的遗传转化。花后12-16天分离未成熟胚，除去胚轴，在粒子轰击之前将未成熟盾片预培养1-2天。金颗粒被含有感兴趣基因和选择性标记基因（在本例中为条形（双丙氨磷抗性））的质粒 DNA 包被，并被发射到细胞中以递送 DNA。随后的组织培养和再生步骤允许恢复幼苗，并通过加入 PPT（草铵膦三肽）（含草铵膦除草剂的活性成分）来帮助选择转化体。这种更新的方法在再生过程的早期引入了选择，从而提供了缩短的方案，同时保持了高转化效率。
The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.