CORTEVA’S EA PROTOCOL

Corteva has published papers12 on using their bacteria Ochrobactrum haywardense H1 to transform soybeans. What I found interesting was the not the species (which I will probably never have access to) but the transformation technique. What they describe is an “embryonic axis” process, similar to this de Melo et al3 paper. We tried the de Melo method and couldn’t get it to work. Even with direct help from the authors. I thought maybe the Corteva papers could give us some pro tips. Unfortunately neither paper has an in depth description of the dissection and preparation, which I think is the important part. They do give recipes for the various media.

Here is the shoot induction media (SIM) recipe from Kumar et al 1

Kumar et al SIM recipe.

Seems like a pretty standard plant tissue culture medium. One interesting point is that there is no 6-benzylamino purine (BAP), which is often found in SIM recipes. The other Corteva paper, Cho et al2 also mysteriously leaves out BAP

Cho et al SIM recipe

Although, BAP is included in their infection medium, which also seems like a strange time to be applying growth hormones. I would assume you would leave things like BAP or GA3 out of the infection medium since it is in contact with the plant tissue for such little time, then add it afterward when you are trying to get organs to initiate and grow. But who knows. As of right now I can’t make the protocol work so maybe my thoughts on soybean tissue culture are incorrect and worthless.

It gets more confusing, though. I looked at one of their patents, US11236347B24, specifically at Table 10 and found they do include BAP in their SIM here. Now I am super confused and have no idea if I need to be adding BAP or not. If it is required then their papers would be non-reproducible. If its not required then they just added it to their patent application for funsies?

SIM from Corteva patent US11236347B2

Like I said earlier, I think my problem is how I am preparing the tissue. That’s normally the problem with these things. But this sloppiness seeds some doubt in my mind about what hormones I should be adding and when.

  1. Cho HJ, Moy Y, Rudnick NA, Klein TM, Yin J, Bolar J, Hendrick C, Beatty M, Castañeda L, Kinney AJ, Jones TJ, Chilcoat ND. Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1. Plant Biotechnol J. 2022 May;20(5):977-990. doi: 10.1111/pbi.13777. Epub 2022 Feb 24. PMID: 35015927; PMCID: PMC9055811. ↩︎ ↩︎

  2. Kumar S, Liu ZB, Sanyour-Doyel N, Lenderts B, Worden A, Anand A, Cho HJ, Bolar J, Harris C, Huang L, Xing A, Richardson A. Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template. Plant Physiol. 2022 Jun 1;189(2):585-594. doi: 10.1093/plphys/kiac075. PMID: 35191500; PMCID: PMC9157123. ↩︎ ↩︎

  3. Paes de Melo B, Lourenço-Tessutti IT, Morgante CV, Santos NC, Pinheiro LB, de Jesus Lins CB, Silva MCM, Macedo LLP, Fontes EPB, Grossi-de-Sa MF. Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration. Front Plant Sci. 2020 Aug 12;11:1228. doi: 10.3389/fpls.2020.01228. PMID: 32903423; PMCID: PMC7434976. ↩︎

  4. US patent US11236347B2 Google Patents ↩︎