Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration

Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration

Melo, Bruno Paes de;Melo, Bruno Paes de;Melo, Bruno Paes de;Melo, Bruno Paes de;Lourenço-Tessutti, Isabela Tristan;Lourenço-Tessutti, Isabela Tristan;Morgante, Carolina Vianna;Morgante, Carolina Vianna;Santos, Naiara Cordeiro;Santos, Naiara Cordeiro;Pinheiro, Luanna Bezerra;Pinheiro, Luanna Bezerra;Pinheiro, Luanna Bezerra;Lins, Camila Barrozo de Jesus;Lins, Camila Barrozo de Jesus;Silva, Maria Cristina Matar;Silva, Maria Cristina Matar;Macedo, Leonardo Lima Pepino;Macedo, Leonardo Lima Pepino;Fontes, Elizabeth Pacheco Batista;Fontes, Elizabeth Pacheco Batista;Grossi-de-Sa, Maria Fatima;Grossi-de-Sa, Maria Fatima;Grossi-de-Sa, Maria Fatima;
Frontiers in plant science 2020 Vol. 11 pp. -
49
melo2020soybeanfrontiers

Abstract

The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employing Agrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity and in vitro tissue 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-cultivation in 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 other Agrobacterium-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.

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