One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

Abstract

Background: Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Results: Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. Conclusions: We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

“…The addition of a selective herbicide in root production medium increased the transformation frequency to 69% (Fig. 3 ) and the process takes only 16 days, faster than other methods [ 14 , 15 ]. Unlike other protocols we used wounded cotyledons as explants, allowing agrobacteria infection better.…”

“…Therefore, using A. rhizogenes to induce transformed hairy roots represents a novel approach in soybean gene functional analysis. Previous reports suggest that hairy roots can be induced from several soybean explant types, such as cotyledon and hypocotyl [14,15]. These transgenic hairy roots have been used to evaluate salt and drought tolerance, nodulation, nematode and pathogen resistance, and virussoybean interactions [7][8][9][10][11][12][13].…”

“…Hairy root transformation systems have been established in soybean to study the metabolism, salt and drought stress, pathogens and nodulation [7][8][9][10][11][12][13]. However, the transformation frequency of current protocols is low and the process takes 30-45 days [14,15]. This hampers the high-throughput characterization of gene function, emphasizing the need for a rapid, simple, and highly efficient procedure.…”

Fluorescent Soybean Hairy Root Construction and Its Application in the Soybean—Nematode Interaction: An Investigation

“…The first step is to use a fresh bacterial carpet because overcultured bacteria will reduce the efficiency [ 18 ]. Secondly, as described in a previous study [ 37 ], bacterial suspensions can be added into vermiculite to enhance the infection efficiency but too much added medium may cause the yellowing of the soybean explant. A high humidity is critical for FHR regeneration.…”

An Efficient Root Transformation System for Recalcitrant Vicia sativa

“…An alternative to regenerating transgenic plants is to produce transformed hairy roots using the bacterium Rhizobium rhizogenes. Examples of success include Glycine max (Cho et al, 2000;Kereszt et al, 2007;Cai et al, 2015;Fan et al, 2020), Vicia hirsuta (Quandt et al, 1993), Vicia faba and Medicago truncatula (Vieweg et al, 2004). Previously, the production of transgenic hairy roots were described in vetch by Tepfer (1990), but no detailed description of the method was included.…”