An Efficient <i>Agrobacterium rhizogenes</i>-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 <i>Agrobacterium rhizogenes</i> is widely applied in studying root-specific processes, nodulation, biochemical and molecular...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-10-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/20/12261 |
| Summary: | The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by <i>Agrobacterium rhizogenes</i> 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 <i>Promoter:GUS</i> 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. |
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| ISSN: | 1661-6596 1422-0067 |