Scalable production of soybean hairy roots: a reliable method for field trials.

key messageWe optimized soybean hairy root production for field trials using newly transformed Rhizobium rhizogenes, a 22 °C growth temperature, and GFP flashlight visualization. Roots are the primary organs for sensing abiotic and biotic stress factors originating in the soil. A critical biological question is whether root plasticity in response to these stresses influences whole-plant development. The generation of soybean chimeric plants with an altered root transcriptome offers a powerful approach to address this question. The existing protocols for this strategy typically require sterile conditions and produce a limited number of chimeric plants. We optimized the technique by introducing several key modifications, significantly enhancing its efficiency. The new protocol eliminates the requirement for sterile conditions in most steps. Moreover, fresh bacterial transformation for each experiment was performed, overcoming the loss of infection ability associated with storing cultures at - 80 °C, reaching 70-100% efficiency. The use of a specific flashlight helped to determine which roots were transgenic, avoiding destructive sampling. A previous transformation of cotyledons helped to select the colony with the highest infection ability. The optimal plant growth temperature was determined to be 22 °C. These combined changes and tips resulted in the consistent production of hundreds of chimeric plants, making them suitable for large-scale field trials. Results from subsequent field trials, performed over three seasons, with different constructs that overexpress or silence transcription factors, and confirmed that root transcriptome alterations impact whole-plant development.