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.

5 thoughts on “Transforming Agrobacterium by the freeze thaw method”

  1. Hi Rahul
    My plates giving a smeared look as if cream is smeared
    I ruled out contamination. Is this due to taking excess quantity and plating

    1. Kamesh,

      It is not real easy for me to diagnose your problem from the information you provided but I will try. You could send a photo that would really help.

      You can certainly plate the entire transformation on a single plate and will have a visible amount of cells on the plate, however, colonies will only form from transformants if selection is used.

      My plates always have a haze of bacteria from the plating that does not grow further.

      If you are getting a lawn growing you may have contamination with a resistant strain (bacteria with plasmid).

      Also, resistant E. coli can grow very well on the shame media and can out compete Agro. Looking at the cells in a microscope can tell the difference between Agro and E. coli (Agro is much smaller).

      Anyway I am not sure if you have no transformants and are looking at EHA105 that cannot grow or you are getting a lawn of bacterial growth. Send a picture of a plain LB plate and one that you are describing and I may be able to help further.


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