A Rhizobium rhizogenes-mediated transformation method coupling CRISPR/Cas9 gene editing in soybean with high-throughput soybean cyst nematode greenhouse bioassays

Soybean (Glycine max) production is constrained by an array of plant-parasitic nematodes resulting in billions of dollars in yield losses annually. Genetic studies have identified numerous plant and nematode genes potentially involved in soybean-nematode interactions, but functional testing remains a significant bottleneck due to the inability to create precise gene knock outs, and the time, labor, and resource intensive processes of generating stable transgenic soybeans and conducting nematode bioassays. Rhizobium rhizogenes-mediated hairy root transformation is a well-established and efficient method for generating transgenic roots, facilitating gene function studies without the labor-intensive process of whole-plant generation; however, current in vitro and soybean composite plant methods suffer from poor nematode infection rates and/or high levels of variation. The one-step hypocotyl slant cut soybean hairy root transformation protocol for generating composite plants was optimized by testing different R. rhizogenes inoculation methods and modifying the existing protocol steps to improve transformation efficiency. This was then coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing for the seamless transition of composite plants from growth chambers to greenhouses for high-throughput nematode bioassays to assess gene function. The composite plant root systems, when reduced to a single transgenic root suitable for soybean cyst nematode (SCN) greenhouse bioassays, provides a comparable set of composite plants for each treatment for use in bioassays ensuring an accurate comparison of SCN infection phenotypes. The method was validated for SCN bioassays using CRISPR/Cas9 genome editing to knock out the soybean serine hydroxymethyltransferase 08 (GmSHMT08) gene in an SCN-resistant genotype. The utility of the protocol for generating transgenic hairy roots was demonstrated across several soybean genotypes, facilitating studies in SCN-resistant soybean genotypes typically recalcitrant to Agrobacterium transformation. This study demonstrates a rapid, simple, and efficient soybean ex-vitro hairy root transformation method for generating CRISPR/Cas9 edited soybean composite plants. The improvements reduce space constraints in controlled environments, decrease root system variation, and enable high-throughput bioassays to study gene function in soybean-nematode interactions.