Transforming Agrobacterium by the freeze thaw method

Plant transformation is frequently carried out with the aid of Agrobacterium. Agrobacterium is a naturally occurring bacteria that genetically modifies plants in the wild. If you have ever seen a tree with a large tumor on it, you have seen the work of Agrobacterium. Currently, scientists exploit the genetic engineering capabilities of a domesticated Agrobacterium to transform plants.

This video shows you how to transform Agrobacterium by the freeze-thaw method. Agrobacterium transformation by the freeze thaw method is cheap, fast and efficient. The transformation and recovery steps can be performed in a single tube. Making Agrobacterium competent cells for the freeze thaw method is easy because it is not necessary to capture the cells in log phase growth. Instead, an overnight culture of Agrobacterium can be used to make these competent cells. The freeze-thaw method does not require electroporation apparatus or expensive electroporation cuvettes. The freeze-thaw transformation method is not as efficient as electroporation, however, it always gives more than enough colonies. A written version of the Agrobacterium freeze thaw transformation can be downloaded here. Once you have your desired binary vector in Agrobacterium, you could easily transform Arabidopsis by Agrobacterium floral dip.

Advantages over electroporation

  1. No expensive equipment required.
  2. Very cheap.
  3. Competent cells are made from an overnight culture rather than a culture in log phase growth.
  4. Transformation and recovery are performed in a single tube.

E. coli transformation and plating

Transforming E. coli with plasmids and plating the transformation.

I have found that the Inoue, H., Nojima, H., & Okayama, H. (1990). Gene, 96(1), 23-28. method gives the highest efficiency competent cells and is very easy to do. You can get a protocol for the Inoue method here.