Improvements on transformation efficiency

Overview

New update July 2003
As in any technology for plant transformation, there are multiple factors involved in Agrobacterium-mediated transformation that influence the success or failure of the transfer of gene of interest into plants and their subsequent stable integration and expression. The different factors can affect transformation differently, depending in part on the plant species.

Aspects of transformation that affect success include:

  • Maturity of the plant – as a general rule young plants are easier to transform than old ones;
  • Selected tissue to be transformed;
  • Agrobacterium strain selected for transformation;
  • Extent of time and conditions for inoculation of the tissue with Agrobacterium;
  • Growth of Agrobacterium with respect to the transformed plant cells. If there is overgrowth of Agrobacterium, the chances of regenerating complete plants from the transformed tissue dwindle;
  • Plant tissue necrosis caused by Agrobacterium.

In this section we present patents and patent applications disclosing improvements related to one or more of the factors mentioned above. The inventors were motivated by a need to enhance the efficiency of Agrobacterium-mediated transformation of plants. The selected inventions refer to:

  • Inhibition of plant necrosis caused by Agrobacterium. Novartis (now Syngenta) and the University of Minnesota each have several granted patents and patent applications filed in the United States, Europe and Australia that disclose methods for inhibiting Agrobacterium-induced necrosis. The methods entail heat shock treatment of the plant tissue, chemical inhibitors, and gene products expressed in the transformed plants that inhibit necrosis and enzyme inhibitors.
  • Inhibition of Agrobacterium growth. Overgrowth of Agrobacterium has a negative effect on the survival rate of the transformed plant cells and increases the number of copies of T-DNA inserts in the transformed plant. Nunhems Zaden has granted patents describing the use of auxotrophic Agrobacterium mutants that allow control of their growth by omission of defined vital nutrients. Monsanto has a PCT application describing the use of compounds containing heavy metals and antibiotics to control the growth rate ofAgrobacterium.
  • Reduction of the weight of the transformed explant. Weight reduction of the explant to be transformed facilitates DNA transfer and favors embryogenic callus formation.Monsanto‘s applications filed in United States and Europe discuss methods for reducing the weight of the explant during the co-cultivation period by extracting moisture from the explant.
  • Sonication of the plant tissue. In this improvement, the target tissue is subjected to ultrasound before, during, or after immersion in an Agrobacterium suspension. The Ohio State Research Foundation has a United States patent and a European patent application on this subject matter.
  • Vacuum infiltration of Agrobacterium into the plant. Agrobacterium establishes a more intimate contact with the cells of a plant when subjected to a vacuum environment. The method is applied in planta, thus avoiding both in vitro culture and regeneration steps. The Samuel Robert Noble Foundation and Paradigm Genetics Inc. have patent applications on the use of vacuum to assist the transformation of any plant, monocots and some particular crops with Agrobacterium.

In conclusion,
The granted patents and patent applications discussed in this section are directed to fairly specific methods applied in Agrobacterium-mediated transformation protocols. With regard to these patents and any patents yet to issue, freedom to operate will become an issue only if any one of the particular procedures as claimed is part of a transformation protocol carried out in the countries where the patents have been granted. Remember that claims as filed in patent applications do not have a defined scope and may change considerably as the applications are prosecuted and become granted patents.