Enhancing Agrobacterium-Mediated Soybean Transformation Efficiency with an Auxiliary Solution

Soybean provides oil, protein, and biofuel. Efficient transformation systems are vital for advancing soybean research. Currently, Agrobacterium rhizogenes-mediated transformation is the predominant method for facilitating rapid transformation, producing transgenic hairy roots. However, the limitations of soybean transformation technology primarily originate from its low efficiency and genotype dependency, leaving significant room for improvement in the development more universally applicable and efficient methods. In this study, we explored the enhancement of soybean transformation efficiency through the generation and validation of three reporter vectors (ZsGreen, TdTomato, and Ruby) and the strategic use of Agrobacterium Auxiliary Solution (AAS) containing Silwet L-77 and hormone mixtures. Our findings demonstate that the incorporation of hormone mixtures and Silwet L-77 into AAS significantly improves hairy root transformation rates. Specifically, the combination of hormone mixtures with Silwet L-77 substantially increased both total root and cotyledon transformation efficiencies compared to the control. We also assessed the impact of vector size on transformation efficiency, observing a notable decrease in efficiency with larger vectors such as the Ruby cassette compared to smaller markers like GFP and RFP. Furthermore, our study examined the effects of AAS on the co-transformation rate of two separate vectors, revealing a slight but significant reduction in efficiency compared to single vector transformations. Additionally, we evaluated the role of AAS in enhancing soybean hypocotyl transformation rates in composite soybean plants across various varieties. The results consistently showed an increase in both positive roots and explant efficiencies with the addition of AAS, indicating its broad applicability and effectiveness in soybean transformation. However, significant varietal differences in transformation rates were observed, particularly between "Forrest" and other varieties such as "Williams 82" and "Dongnong 50". In summary, our research emphasizes the significant role of auxiliary agents and vector size in optimizing soybean transformation techniques, providing valuable insights for future advancements in soybean genetic modification and biotechnological research.