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An Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method in a Soybean Root Biology Study

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes...

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Detalles Bibliográficos
Autores principales: Huang, Penghui, Lu, Mingyang, Li, Xiangbei, Sun, Huiyu, Cheng, Zhiyuan, Miao, Yuchen, Fu, Yongfu, Zhang, Xiaomei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9603872/
https://www.ncbi.nlm.nih.gov/pubmed/36293115
http://dx.doi.org/10.3390/ijms232012261
Descripción
Sumario:The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.