What is the present white paper about?

This white paper on Agrobacterium -mediated transformation of plants explains the basic scientific aspects of transformation as well as the key intellectual property aspects of methods and materials used in transformation.

This paper has been expanded to encompass transformation of organisms outside the plant realm. Patents directed to the transformation of fungi and algae are part of the new additions as well as patents related to improvements on plant transformation efficiency. The latest version of the paper is organized into the following 12 sections:

Introduction
The introduction first explains what the CAMBIA intellectual property resource intends to accomplish in this white paper and then provides brief summaries of each of the seven main sections of the paper. Importantly, the introduction informs you of some of the topics and subject matter areas you will not find analyzed within but that may still be important for obtaining freedom to practice some of the inventions described in this paper.

Because many web sites, workshops, and pamphlets that describe basic intellectual property principles (e.g., what is a patent; the requirements and standards for obtaining a patent) are widely available, we do not duplicate those efforts here. We do present, however, as a companion tutorial, guidelines on “How to read a patent”. In addition, some key facts about patents that are often overlooked or forgotten by newcomers to patent literature are emphasized in the introduction. It is our belief that familiarity with these concepts will assist you in navigating the sometimes murky waters of patents.

Scientific aspects
This section provides some historical perspective and basic scientific information regarding Agrobacterium-mediated transformation of plant cells. The structure and use of two basic types of vectors, co-integrated vectors and binary vectors, are discussed.

The patent information in the following sections comprises an overview, a summary page presenting the key issues raised by the patents and patent applications (illustrated by comparing them and pointing out the most limiting aspects of the claimed inventions), and provides detailed information on each patent and patent application including bibliographic data, a summary of the claimed invention and independent claims.

Types of tissues to be transformed
Agrobacterium infects some tissues more efficiently than others. Reflecting this variability, specific protocols have been developed for different tissue types. Some of these methods have been patented, and it is these patents that are discussed in this section. The patents are generally directed to transformation of callus, immature embryo, pollen, shoot apex and live plants.

Binary vectors
Binary vectors are the major vector system used in Agrobacterium-mediated gene transfer. The binary vector system comprises two independent and complementing vectors: one vector having a T-region and the gene of interest and the other vector having a vir region. Two sets of patents and applications are presented and analyzed. The first set is directed to basic vector designs and methods of constructing them. The second set is directed to special applications using these vectors or improvements on the basic vector design.

Co-integrated vectors
Although historically the first vector system to be developed, co-integrated vectors are less widely used. In this system, a recombined vector is constructed from a Ti plasmid and a small plasmid containing a gene of interest between two T-DNA borders. The patents and applications in this section are directed to the basic forms of the vectors, including the primary elements of the plasmids, and to basic methods for assembling the recombined, co-integrated vector. Additionally, a set of patents and applications is discussed that claim improved vector design and methods for their use.

Mobilisable vectors
This new system of vectors appears to be an alternate system to the binary and co-integrated vectors systems. The plasmids used in this system are derived from plasmids belonging to the family Enterobacteriaceae (e.g., E. coli). They are non-conjugative plasmids, thus, they are not able to transfer by themselves into a cell host as derived Agrobacterium Ti-plasmids are able to do. Mobilisable plasmids require the presence of a helper plasmid that supplies the transfer genes required for the transformation of the host cell. In addition, a gene of interest is not surrounded by T-DNA borders in a mobilisable plasmid. Although there is currently (September 2001) only a European application related to this vector system, we present it here as an alternative to the crowded patent landscape of the traditional vector systems.

Improvements on transformation efficiency
There are multiple protocols for Agrobacterium-mediated transformation that vary according to the tissue to be transformed, the plant and the purpose of transformation, among other reasons. Improvements of transformation efficiency can be gained by using compounds to control the growth of Agrobacterium and the undesired effects of tissue browning, as well as by using physical procedures to facilitate the inoculation of the bacterium into the host plant. The patents in this section are directed to methods for improving transformation efficiency and include methods of controlling Agrobacterium growth, inhibiting necrosis of the transformed plant tissue, reducing the weight of the explant to be transformed and applying physical treatments, such us sonication of the plant tissue and vacuum infiltration, to promote the intimate contact between the bacterium and the host plant cell.

Monocot transformation
The world of flowering plants with protected seeds (Angiosperms) is sometimes neatly divided into monocotyledonous (monocot) and dicotyledonous plants. Most of the important staple crops of the world, that is, cereals, are monocots. Initially it was difficult to transform monocots using Agrobacterium, but eventually this constraint was overcome. Several key patents were awarded to the entities able to accomplish this feat. The patents discussed in this section include those broad patents directed to transformation of any monocot as well as patents directed to transformation of any cereal plant (e.g., wheat, barley, rice, maize) and to transformation of a particular individual monocot plant (e.g., banana, pineapple, rice, sorghum).

Dicot transformation
The second major classification of flowering plants with protected seeds (Angiosperms) is dicotyledonous plants (dicots). Early on, dicots were readily transformed by Agrobacteriumand so in general, there are fewer patents in this area. Following a presentation of the patents directed to general transformation methods, which generally are limited to the use of co-integrated vectors or binary vectors, patents and applications directed to particular dicot species are presented. Some of these particular dicots are beans, cacao, cotton, peas, roses, soybean, and tomato.

Conifer transformation
Non-flowering plants with naked seeds that appear in a cone are called Gymnosperms. Conifers are the largest group of plants within the Gymnosperms. Conifers such as Pines are very important as a source of timber for construction and for paper pulp. Several chemical compounds extracted from pines are used in the pharmaceutical, cosmetic and food industries. For many years, Agrobacterium-mediated transformation of conifers was deemed impossible but the barriers for their transformation have been overcome. Patents on this area describe several methods to attain transformation of pines.

Marine algae transformation
Algae are organisms found in virtually every ecosystem, in ecosystems as diverse as marine, freshwater and terrestrial habitats. Algae are commercially very valuable. For example, marine algae or seaweeds are used in many maritime countries as a source of food, for industrial applications and as a fertilizer. Marine algae’s products such as gums are very important in the international market. Although Agrobacterium-mediated transformation of eukaryotic organisms was initially confined to plants for a while, nowadays, algae can also be transformed via this bacterium. Because transgenic marine algae with a large biomass are a potential source for valuable pharmaceutical and industrial products, patent activity in this area will possibly increase. Currently, there is a patent application directed to methods for transforming multicellular marine algae.

Fungus transformation
Fungi constitute a separate life kingdom from animals and plants. Most fungi are filamentous organisms that contain two nuclei per cell for most of their life cycle. Fungi are essential organisms required for the continuous cycle of nutrients through ecosystems. While they provide essential nutrients to vascular plants through symbiosis, not all of their activity is beneficial. In this regard, many fungi are the cause of plant, animal and human diseases. The selected patents on Agrobacterium-mediated transformation of fungi are mainly directed to the transformation of filamentous fungi, commonly known as moulds. Transformation of yeasts, another group of fungi, is outside the scope of this paper.