Types of Tissues to be transformed

Summary

The efficiency of T-DNA transfer via Agrobacterium to a plant varies considerably, not only among plant species and cultivars, but also among tissues. Various protocols for Agrobacterium-mediated transformation of plants use leaves, shoot apices, roots, hypocotyls, cotyledons, seeds and calli derived from various parts of a plant. In other methods, the transformed tissue is not removed from the plant but left in its natural environment, thus, the transformation takes place in planta.

Patents directed specifically to methods of transforming different tissues are relatively few, but the scope of their protection is rather broad. Some of the patents referred to in this section are considered key patents for widely used technologies by the research community.

The patents discussed in this section are directed to the transformation of callusimmature embryopollen, seed, shoot apex parts in culture as well as in planta . With the exception of Japan Tobacco‘s patents directed to callus and immature embryo transformation of a monocotyledonous plant, claims in these patents are not restricted to the type or species of plant to be transformed. Therefore, any plant arguably falls within the scope of the claims of these patents. The bacterium used for transformation is Agrobacterium or specifically A. tumefaciens.

  • Callus transformationJapan Tobacco has two granted patents, one in Australia and the other in the United States. The Australian patent claims a method of transforming a monocot plant tissue with Agrobacterium.  The plant tissue can be from any portion of any type of monocot plant and is either already dedifferentiated or is exposed to a dedifferentiation process. In the United States patent the tissue to be transformed must be not less than 7 days old.
  • Immature embryo transformation.  Japan Tobacco also has a patent in Australia claiming  transformation of the scutellum of an immature embryo of a monocot plant with Agrobacterium. The transformation process takes place before the tissue has differentiated into a callus.
  • Pollen transformation. A patent related to this topic was granted to the United States Department of Agriculture (USDA) in the US and a patent has also been granted in Australia. In these patents, transformed pollen fertilizes a second plant to obtain transgenic seed, which is germinated to obtain a transgenic plant.
  • Shoot apex transformation. Transformation of an excised shoot apical tissue by inoculating the tissue with A. tumefaciens is disclosed by Texas A & M University in a granted United States patent. Applications filed in Europe and in Australia have been abandoned.
  • In planta transformation. Three different entities have filed patent applications on in planta transformation. Cotton Inc.Rhobio and Performance Plants‘ patent applications refer to transformation of a plant tissue with Agrobacterium in its natural environment. Performance Plants’ patent applications, however, were recentlyabandoned. In contrast to Rhobio, which does not claim a method of transforming a particular tissue of a plant, Cotton Inc. claims the injection of Agrobacterium into floral or meristematic tissue. Furthermore, Cotton Inc. claims appear to require the transformed plant cells or tissue to develop and regenerate within the plant, whereas  Rhobio‘s patent claims are directed to removing tissue from the plant and regenerating it in vitro.
  • Floral transformation (Update July 2003) is basically an in planta method that has become very popular in the transformation of Arabidopsis thaliana (Brassicaceae), one of the best known model plants in genomic studies. It is also suitable for the transformation of monocotylenous plants. A US patent assigned to Rhone-Poulenc Agro and a PCT application assigned to Paradigm Genetics Inc. are described in the section ” General Transformation Methods for Monocots.” A US patent granted to Cotton Inc.which discloses transformation of floral or meristematic tissue (mentioned in the preceding paragraph) is discussed in the section “In planta transformation.”  A patent has been granted to Paradigm Genetics Inc. in which a new method to transform plants by direct treatment of flowers is described. The method is based on published literature and represents a simple modification to the adjustment of cell density of the Agrobacterium strain used in the transformation: cells are diluted rather than centrifuged.
  • Seed transformation (Update July 2003). Two groups have filed patent applications on transformation of plants using seed as target tissue. The Agri-Biotechnology Research Center of Shanxi (China) has filed a US and an EP application based on a Chinese patent application. As filed, these two applications contain general claims to applying Agrobacterium to germinating seed, with no further treatment of the seed. A PCT application has also been filed by Scigen Harvest Co Ltd from Korea on a method using needle-wounded seed as target tissue in combination with Agrobacterium tumefaciens.

In conclusion

  • Transformation of pollen with Agrobacterium is fairly broadly protected in the United States and in Australia. The situation is similar with shoot apex transformation in the United States, except that the bacterium used in this case is specifically A. tumefaciens. Thus, use of other species of Agrobacterium to transform apical shoots from any plant may fall outside the scope of the claimed invention.
  • There appears to be more room to avoid infringing patents on in planta and callus transformation. Although other entities may have applications pending, only Cotton Inc. has been granted a United States patent, which particularly claims:
    1. transformation of floral or meristematic tissue, and
    2. the use of a needleless device to inject Agrobacterium into the tissue.

Thus, if one of these two elements is not part of an in planta transformation process, the process may be well outside the scope of the claims of Cotton Inc.’s patent.

With respect to callus transformation claimed by Japan Tobacco, at least in the United States, the tissue must be at least seven days old. Thus, if tissue can be used that is less than 7 days in culture, literal infringement of this patent may be avoided.

  • Patent applications for the transformation of the tissues mentioned above are pending in Australia, Europe, United States and some other jurisdictions and may become an issue for freedom to operate if they are granted as filed or may be prior art for other inventions related to the Agrobacterium transformation of these particular tissues.
  • Transformation of germinating seed with Agrobacterium will be protected broadly if pending applications are granted, especially the one to The Agri-Biotechnology Research Center of Shanxi.