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

Abstract

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.

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY

“…Chen et al (2018) have reported different percentages of hairy root induction (90-99%) and transformation efficiency (30-60%) in Williams 82, Jack, Zigongdongdou, Heihe 27 and Zhonghuang 30 cultivars using strain K599. In another study, transformation efficiencies of 62.7% and 70.1%, respectively, have been reported for Williams 82 and Tianlong 1 using strain K599 (Huang et al, 2022). Here, we also tested different plant genotypes, across different A. rhizogenes strains, and observed a significant effect of the genotype on in vitro hairy root induction and transformation efficiency.…”

“…With the same A. rhizogenes strain, the optimum percentage of hairy roots can be influenced by the infection period (inoculation duration) and A. rhizogenes cell density (Boobalan and Kamalanathan, 2020). This is despite previous studies optimized in vitro hairy root transformation of soybean with a constant level of these parameters (OD 600 = 0.6/30 min) (Chen et al, 2018;Cheng et al, 2021), (OD 600 = 0.8-1/30 min (Huang et al, 2022). In the first in vitro experiment of the present study, the effects of different A. rhizogenes cell densities and inoculation durations on hairy root transformation were evaluated using an orthogonal experimental design.…”

“…Although K599 is the most common strain used to optimize in vitro hairy root induction in soybean (Chen et al, 2018;Cheng et al, 2021;Huang et al, 2022), testing different strains and finding the most effective one is necessary, as hairy root stimulation and gene transfer efficiency are directly influenced by the A. rhizogenes strain used (Thwe et al, 2016) and differential efficiencies of A. rhizogenes strains have been reported in hairy root induction of other plants such as African geranium (Yousefian et al, 2021), fenugreek (Tariverdizadeh et al, 2021), tobacco (Qin et al, 2022;Yektapour et al, 2022) and marshmallow (Tavassoli and Safipour Afshar, 2018). In this study, the effect of A. rhizogenes strains was significant on in vitro hairy root induction percentage and transformation efficiency.…”

The Glycine soja cytochrome P450 gene GsCYP82C4 confers alkaline tolerance by promoting reactive oxygen species scavenging

“…Agrobacterium‐mediated transformation is an effective method to obtain transgenic chimeric plants including the economically important crop soybean (Zhu, 2016; Huang, et al 2022). Studies showed that overexpression of GmCAMTA12 and GmCIPK2 in transgenic soybean plants with hairy roots exhibited tolerance to drought stress (Noman, et al 2019; Xu, et al 2021), while the GmPKS4 are highly resistant to salt and saline stress (Ketehouli, et al 2021).…”

Enhancing Agrobacterium-Mediated Soybean Transformation Efficiency with an Auxiliary Solution

“…Soybean transformation, surpassing traditional breeding methods, plays an indispensable role in addressing fundamental biological queries(Yamada et al 2012; Jin et al 2021; Huang et al 2022a). Despite the development and refinement of soybean transformation systems over the past three decades since their initial introduction(Yun et al 2022; Zhai et al 2022; Li et al 2023), achieving stable genetic transformation in soybean remains less efficient compared to other crops(Cheng et al 2021).…”