Abstract
BackgroundAgrobacterium 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.ResultsHere, 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.ConclusionsWe 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.
Highlights
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 reliability of the one-step ARM transformation method was validated by CRISPR/Cas9-mediated knockout the resistance to nodulation gene Rfg1 in the soybean genotype Williams 82 (Nod-) inoculated with Sinorhizobium fredii USDA193 [20, 21]
Rhizogenes strain K599 was used because the hairy roots can be effectively induced in soybean [11, 23]
Summary
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 hairy roots were induced in a medium, and the generated composite plants were transplanted into another medium for growth This two-step operation is time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Agrobacterium rhizogenes-mediated (ARM) hairy root transformation offers a powerfully transient tool for investigating root biology and has been widely established in a variety of plant species, including the stably genetic transformation-recalcitrant plant species, such as soybean [10, 11]. The induction of hairy roots and the growth of composite plant were finished in a single growth medium This is a promising technical breakthrough in soybean ARM transformation with simpler operation, higher transformation efficiency, and lower contamination risk in the study on soybeanmicrobe interactions. We anticipate that the one-step transformation method will be widely applied to analyze root-related events in tomato and other plant species besides soybean
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