Soybean Embryonic Axis Transformation: Combining Biolistic andAgrobacterium-Mediated Protocols to Overcome Typical Complications ofIn VitroPlant Regeneration

The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employingAgrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity andin vitrotissue culture led to the development of other DNA delivery systems, such as the biolistic method. Herein, we developed a new one-step protocol for transgenic soybean recovery by combining the two different transformation methods. This protocol comprises the following steps: agrobacterial preparation, seed sterilization, soybean embryo excision, shoot-cell injury by tungsten-microparticle bombardment,A. tumefaciens-mediated transformation, embryo co-cultivationin vitro, and selection of transgenic plants. This protocol can be completed in approximately 30-40 weeks. The average efficiency of producing transgenic soybean germlines using this protocol was 9.84%, similar to other previously described protocols. However, we introduced a more cost-effective, more straightforward and shorter methodology for transgenic plant recovery, which allows co-cultivation and plant regeneration in a single step, decreasing the chances of contamination and making the manipulation easier. Finally, as a hallmark, our protocol does not generate plant chimeras, in contrast to traditional plant regeneration protocols applied in otherAgrobacterium-mediated transformation methods. Therefore, this new approach of plant transformation is applicable for studies of gene function and the production of transgenic cultivars carrying different traits for precision-breeding programs.