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

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.

Keywords: Agrobacterium-mediated transformation; Glycine max; embryonic axis; genetic transformation; high-efficiency plant transformation; particle bombardment.

Figure 1

Biolistic and Agrobacterium-mediated soybean embryonic axis transformation workflow. The workflow of the proposed protocol in this study consists of a one-step soybean embryo infection followed by plant regeneration to obtain transgenic soybean plants. The workflow is divided into three main parts that are followed day-by-day until the plant regeneration step, which requires up to 6 weeks until seedling acclimation. The protocol starts with isolation and bacterial culture (A) in parallel with seed sterilization and hydration (B). The next step achieves the isolation of embryonic axis (C), bombardment of shoot cells that will be transformed and regenerated into a reproductive plant, (D) and, finally, A. tumefaciens (GV3101 strain) infection (E). After embryo infection, cocultivation occurs (F) and is immediately followed by plant regeneration (G) until the acclimation step and plant recovery (H).

Figure 2

Tungsten-coated carrier membrane preparation. (A) Carrier membrane (24 mm diameter); (B) Rupture disk (13.2 mm diameter); (C) Metallic ring support of carrier membrane; (D, E) Distribution of prepared tungsten microparticles at carrier membrane surface; (F) Prepared tungsten-coated carrier membranes under hood to drying. All manipulation in tungsten-coated membranes’ preparing may be performed under pre-cleaned and UV-sterile hood.

Figure 3

Embryonic axis excision and shoot exposing. (A) Pick one hydrated soybean seed and with help of a tweezes and a scalpel make a small section in the opposite of hilo; (B) Discard the embryo non-associated cotyledon and (C) carefully detach the embryo axis with scalpel. (D) Remove the primary leaves to expose shoot cells; (E) Organized embryo in CCM to bombardment.

Figure 4

Soybean embryo axis bombardment. (A) Isopropanol-soaked rupture disks attached to the tip of helium pressure chamber; (B) Protective metallic net at the support of carrier membrane; (C, D) Carrier membrane attached to ring support at holder in bombardment chamber; (E) Embryonic axis at CCM plate into bombardment chamber.

Figure 5

Agrobacterium tumefaciens infection, co-cultivation and plant regeneration. (A) Bombarded embryos in Agrobacterium suspension (left) and after washing (right), performed after 40 min of infection; (B) Infected embryos at CCM. The duration of co-cultivation step at CCM is 16 h; (C) Infected embryos at DRM after co-cultivation in CCM. The plant-regenerative process starts as from the transference of infected embryos to DRM. The A. tumefaciens is not inactivated by chemical treatment and the infection continues along with plant regeneration. At this point, plant surveying is essential to avoid contamination, which should be eliminated by transferring the plant to a new medium or by chemical treatment, if necessary; (D, E) Shoot- and root-regenerated seedlings after 3–6 weeks in DRM. (F) After complete seedlings’ regeneration, the plants can be acclimated in standard greenhouse protected with a plastic bag for 1 week and follow normal cycle of development until seeds recovery. All steps of plant characterization can be performed at this point.

Figure 6

Regenerated plants under full development in greenhouse. (A) In vitro recovered seedling after DRM-regeneration and 1-week of plant acclimation. (B) Putative transgenic T0-matrixes in vegetative stage. (C) T1-recovered plants in reproductive stage.