Department of Chemical Engineering, Northeastern University, Boston, MA, USA.
Department of Biology, Northeastern University, Boston, MA, USA.
Methods Mol Biol. 2022;2469:129-144. doi: 10.1007/978-1-0716-2185-1_11.
Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.
发根农杆菌是引起双子叶植物毛状根病的细菌，并被专门设计用于开发转基因毛状根培养物。由于其遗传和代谢稳定性，毛状根培养物作为研究转基因功能的组织培养系统和作为特殊代谢物或蛋白质生产平台具有优势。生成毛状根培养物的过程包括首先用发根农杆菌感染外植体，从新出现的毛状根中切除并消除发根农杆菌，在含有选择剂的平板上选择转基因毛状根，通过 PCR 确认转基因的基因组整合，最后在液体培养基中适应毛状根。在这里，我们提供了一个详细的方案，用于开发和维持我们感兴趣的药用植物长春花的转基因毛状根培养物。
