Agrobacterium-mediated transformation - Overview

Many methods and techniques can be used to transfer genes into cells. In agricultural plant biotechnology, the most widely utilized technique is Agrobacterium-mediated transfer, which is heavily patented.  Use of patented technologies can restrict the deliverability of products. 

About this technology landscape

• This landscape was originally authored by Carolina Roa-Rodriguez with the assistance of Dr. Carol Nottenburg.  It was updated in 2003 by Dr. Jorge Mayer.   Further updating of some sections was done in 2005-2006 by Dr. Shoko Okada with the assistance of Dr. Marie Connett Porceddu and Dr. Dianne Rees.
• Funding for this project was provided by the Rockefeller Foundation.  The web version was originally produced by Doug Ashton and we are grateful for web production assistance for the updates by Dr. Nick dos Remedios, Steve Irwin, and Annet Maurer.  

Introduction: Why an IP and Technology landscape on Agrobacterium-mediated transformation?

In our experience, the intellectual property landscape in biotechnology areas is often not very well understood by the research community, especially the public sector. All too often rumours and misstatements about patents are passed along from researcher to researcher. This is an unfortunate situation, however understandable. But with the increasing importance and emphasis on patents, it is becoming necessary for scientists to be versed in the field of intellectual property.

With this paper and others now present on or planned for the Patent Lens, we strive to provide a readable and understandable overview of patents in some key areas of biotechnology. In this way, we hope to contribute to the public awareness of intellectual property issues that surround these key biotechnological tools. The information in the white papers is not exhaustive, but consists of selected documents found to broadly encompass the area. To satisfy the myriad questions and issues raised by the research or the interests of each person who visits this site would require a host of attorneys and an enormous amount of time. Instead, this paper is provided in order to open the door into the patent world and furnish platform knowledge from which additional self-directed investigation can be performed.

This first technology landscape is focused on the intellectual property concerning methods and materials used for Agrobacterium-mediated transformation of plants. This transformation method is currently one of the most widely used means of making transgenic plants. Although much of the basic research and findings that led to Agrobacterium-mediated transformation was done in public institutions, the private sector now holds many of the key patent positions. The patents were obtained by the private sector either from internal research and development or from public institutions in the form of a license or occasionally as the assignee. Thus, the science and the patent positions are of high interest to both public and commercial sectors.

Technology landscapes, by their very nature, become outdated.  While this landscape contains much useful information about the broad state of the art at the time broad patents were issued (which is critically important to evaluate the ongoing constraints to use of Agrobacterium technology), some patents have lapsed and others have come into force.  The version you see here starts with a list of the updates to this landscape done in 2003, and pages updated since 2003 show the dates of new searches.  But as sections of this landscape would need constant updating, an impossibility with CAMBIA's small team, we welcome updates and inputs by others through the comments interface available on every page of this version of the technology landscape. 

For a way around the Agrobacterium patent morasse, see CAMBIA's Transbacter project.

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 Agrobacterium and 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.

What is the present white paper NOT about?

To learn more about patents and patentability, please visit our companion tutorial, "How to read a patent" and web sites such as the web site of the United States Patent Office and the web site of the World Intellectual Property Organization. Other resource sites may be found on the Links page.

The user should especially note that the materials provided in this site are not comprehensive. In particular, we do not analyze patents directed to methods of using or transforming eukaryotic cells or components of eukaryotic or bacterial vectors that are also used in agricultural R&D. Some of these patents may dominate the agricultural patents discussed on this site. As well, we present only a selected set of patents and applications. The set represents what we consider to be key in the field. It is inevitable that others would have a different opinion about what is key and, as a result, may well have chosen a different set of patents.

This white paper presents an overview of the field of Agrobacterium- mediated transformation with respect to intellectual property. The reader should gain an appreciation for the complexities of the field and insight into the types of intellectual property directed to this field.

What you NEED to know about patents

Claims define what is patented

It is the claims that define the boundaries of the patent owner's rights. Remember that the patent owner's rights are exclusionary: she may exclude others from making, using, selling, offering to sell, and importing the patented invention (e.g., a product or a process) and importing a product made by a process patented in the importing country. To determine if someone is infringing a patent, that is making, using, etc., without the patent owner's permission, the allegedly infringing product or process is compared only to the claims.

Don't fall into the trap of concluding that the title or the abstract or the general description found in the text of the patent indicates what is patented. For example, United States Patent No. 6074877 is titled "Process for transforming monocotyledonous plants". From the title, it sounds like these patent owners have protected a transformation process(es) for transforming all monocot plants. Examination of the claims shows, however, that only transformation of cereal plants is protected, and furthermore, that the method involves wounding an embryogenic callus or treating an embryogenic callus with an enzyme that degrades cell walls prior to transferring DNA into the cells with Agrobacterium. A bit different from what the title implied.

Yet, claims cannot to be interpreted in a vacuum. Although claims define the invention, the scope of the claimed invention is not always clear from reading the plain language of the claim. Claim interpretation can be difficult; a proper analysis is done by reading the claims in the context of the specification and in the context of the "prosecution history" (the back and forth negotiations between the patent applicant and the patent office regarding the claim language). In the case above, for example, several terms in the claims (e.g., "cereal plants", " embryogenic callus", and "enzyme that degrades cell walls") are unclear without additional insight hopefully provided by the specification and prosecution history.

Claims in this white paper and the claims written in "plain English" were analyzed from the plain language and the specification. The prosecution history was not examined. Thus, scope of the claimed inventions may not have always been precisely determined.

A patent application is not the same as a patent

During the application process, patent specifications are published 18 months after the earliest filing. The publications contain the claims as filed. Sometimes the claims are written much more broadly than is actually patentable. As the application is examined by a patent office and claim language negotiated, the claims may shrink in scope. In contrast, the specification of a granted patent will usually be the same as when filed; new matter is not allowed to be added to the text after it is filed.

Because the claims in an application are what the applicant hopes for and not what she will necessarily receive, it is important to know whether you are looking at a granted patent or a patent application.

How do you tell the difference between a granted patent and a patent application? Although every country uses its own system of identifying granted patents, some general guidelines will assist you for the major jurisdictions.

• United States: until 29 November 2000, all publications were issued patents. Currently, the United States identifies patents with a 7-digit number followed by a B1 (indicates a patent not previously published) or a B2 (indicates a patent previously published). E.g., US 6,174,724 B1, shown below.
  
  
  
  
  Patent applications are indicated with the year as a 4-digit number and a publication number followed by an A1 (for the first publication), A2 (for republication) or A9 (corrected publication). E.g., US 2001/0002490 A1, shown below.
  
  
• Europe: patents are indicated with a 7-digit number followed by a B1, e.g., EP 0 458 846 B1 (shown below). A B2 number indicates that the claims have been modified after grant.
  
  
  
  
  Patent applications use the same numbering system but the number is followed by A1, A2, etc., e.g., EP 0 955 371 A1 (shown below).
  
  
• World Intellectual Property Organization (WIPO): often simply called PCT (Patent Cooperation Treaty) applications, publications from WIPO are only patent applications. The publication numbers have a WO, which stands for "world" followed by the year as 2 digits, followed by a publication number, and A1 (first publication), A2 (second publication), etc. e.g., WO 00/34491 A2 (shown below).
  
  

The truth about international patents

A patent is awarded by the government of a country and is valid only within its territorial boundaries. To obtain a patent that is valid in a particular country, a request must be made in that country's patent office.

The confusion and misunderstanding about "international patents" arises sometimes from the PCT process of pursuing patents. When looking at a PCT application, many people erroneously, but understandably, conclude that it is an application for a patent that will be valid in multiple countries. Indeed on the front page of a PCT application (presented below), in the upper right corner there is a heading titled "Designated states" followed by a list of two letter codes. Each of those codes stands for a country (e.g., AU, Australia; CA, Canada; CN, China, and so on). There can be as many as about 110 countries listed. However, this list does not mean that the application is a patent, or even will become a patent, in all of these countries.

OK then, what does this list mean? Through an international treaty (Paris Convention Treaty), a group of countries agreed to not discriminate against each other by affording patent applicants in these countries a one-year period in which to file an application in one of the other countries without losing the benefit of their filing date. The advantage is that any "art" that became known after the original filing date in the home country but before the filing date in another country could not be cited against the application. Thus, for example, if you originally file an application for your invention in Canada, you could wait up to one year before filing the application in Mexico. This would give you time to see if the costs of filing in other countries is justified.

Later, a second treaty (Patent Cooperation Treaty (PCT)) established another route to delay the additional filings in other countries. In this method, an international office was set up (World Intellectual Property Office (WIPO)) to receive and process the applications. But now, the applicant has one year to file at the WIPO office and by designating member countries she preserves her rights and original filing date in those designated countries without having to go to the expense of actually filing in each country. This saves an enormous amount of money! Eventually to obtain a patent in these countries, the application does need to be filed in the national patent offices (the process is called "conversion"), pay fees, have translations done and comply with the regulations of each individual office. Depending on some procedural issues and fee payments, the applicant has either 20 months or 30 months from the original filing date (the date the application was filed in the home country) to file in each of these other countries. Given the costs, most applications are filed in a few other countries at most.

What is ownership of a patent

The legal owner of a patent is designated as the "Assignee" on United States patents and as the "Applicant" on patents in the rest of the world. However, the rights of a patent holder are like a bundle of sticks, and only one of the sticks is legal ownership.

Patent law gives the patent owner the right to exclude others from making, using, offering for sale, selling, and importing the patented product and from using the patented process, as well as using, offering for sale, selling, or importing a product obtained directly from a patented process. These rights are tradeable. The typical form of trade is a license, in which some or all of the rights may be transferred. For example, the patent owner may license only some of the claims in a patent, all of the claims but only in a particular field of research, all of the rights but only in certain countries, or the right to make and use but not the right to sell. Other types of licenses may also be granted.

Unlike the ownership of a patent, which is a matter of public record, licenses can be private. Unless the parties to a license choose to reveal the relationship, it is impossible to know about.

In this paper, the legal owner is noted. The cautionary note is that the legal owner may not be the party that is in control of the rights you want access to.

Preface

Except where otherwise noted, patent information is current through to January 2003.

Summary

Both granted patents and pending patent applications are subject to change.  A granted patent is typically in force for a 20 year term, calculated from the filing date, as long as the maintenance fees are paid, although some patents have been issued under rules that give them different terms (see tutorial).

An example of a patent that has taken advantage of a pre GATT-TRIPS filing date is US Patent No. 6051757. For this patent, the filing date was June 5, 1995; shortly before the June 8, 1995 GATT/TRIPS deadline in the United States.

US Patent No. 6051757 is a continuation application that claims priority to a parent application with a January 14, 1983 filing date. Had this application been filed three days later, it would have likely had a patent term that expired on January 14, 2003.

However, since the application was filed before the GATT/TRIPS deadline, it is entitled to a patent term calculated 17 years from the issue date, rather than 20 years from the priority date.

The application data provided by PAIR reveals that this application took nearly five years after the filing date to issue. Numerous extensions of time were granted by the examiner during the prosecution of the application. This patent finally issued on April 18, 2000, and is therefore likely entitled to a patent term that would expire on April 18, 2017 (barring any litigation or lapses due to failure to pay maintenance fees). Basically, an invention that was made in 1983 gave rise to a patent that expires 34 years after the first filing date!

The patent term is a period during which the patentee has the right to exclude others from using the technology.  Technology described in a granted patent that lapses due to lack of payment or expiration of the term moves into the public domain, and unless the technology is covered by other patents still in force, people may work inventions in the public domain without infringement.

From the moment of filing, patent applications go through an interactive process between the applicant and the patent office, the so-called "prosecution", which eventually leads to the grant or rejection of a patent application. During this process, which may take several years, the claims, which define the scope of desired protection for the invention, are likely to be amended. Therefore, the claims of a published patent application may differ from those finally granted by a patent office. In addition, an application may be abandoned along the examination process if the applicant decides not to seek patent protection for the invention in a particular country.

This white paper on Agrobacterium-mediated transformation of plants was updated in March 2002 and June 2003, and is undergoing another revision now. The dynamic nature of intellectual property rights, especially in a rapidly evolving area such as biotechnology, makes regular updates necessary in order to keep abreast of new constraints to freedom to operate or of formerly patented technology that becomes freely accessible.

The main changes registered are:

• Abandonment, when the applicants have decided not to continue with the prosecution process or when maintenance fees have not been paid for granted patents;
• Issuance of a patent, when applications listed in previous versions of the paper have resulted in granted patents, which may have different claims;
• Entering European (EP) phase, or national phase in other national patent offices (applications filed under the Patent Cooperation Treaty may be converted to national patent applications after a maximum period of 30 months from the earliest priority date).

A summary table provides information on changes between 2002 and 2003. For convenience, the documents are presented according to the white paper sections to which they pertain and following the order set in the table of contents/index of the document. You will find out more detailed information by following the links provided for each patent application.

• New patents and patent applications

Many new patents and patent applications have emerged in the field of Agrobacterium-mediated transformation since 2002. Some of these patents are directed to new methods for transformation of plant tissues and crops, previously discussed in the white paper, and others are directed to new crops, such as coffee, onions, turfgrass and woody tree species.

The new patent documents are presented in a summary table. Documents are grouped according to the white paper sections set in the table of contents/index. You will find out more detailed information on each patent document by following the links provided in the table.

Changes in legal status of patents and patent applications since last update

New patents and patent applications (Update July 2003)

Note! Assignees listed in brackets are assumed (from related applications and patents), because the assignee is often not recorded on US applications.

Assignees in parentheses are assumed, based on related applications and patents, because they usually don't show on US applications

Scientific Aspects

Overview

Agrobacterium-mediated transformation of plants: from a naturally occurring nuisance to a major tool for plant transformation

Agrobacterium tumefaciens is a common soil bacterium that naturally inserts its genes into plants and uses the machinery of plants to express those genes in the form of compounds that the bacterium uses as nutrients. In the process, Agrobacterium causes plant tumors commonly seen near the junction of the root and the stem, deriving from it the name of crown gall disease. The disease afflicts a great range of dicotyledonous plants, which constitute one of the major groups of flowering plants.

In 1907, the bacterium was identified by Smith and Townsend as the causative agent of the disease, but it was not until the end of the sixties that a correlation between the tumor and the presence of genetic material of the bacterium was established (Braun and Schilperoort).

Between the 1970s and 1980s, some striking aspects were discovered about the biology, biochemistry, and molecular biology of Agrobacterium. Tumorous plant cells were found to contain DNA of bacterial origin integrated in their genome. Furthermore, the transferred DNA (named T-DNA) was originally part of a small molecule of DNA located outside the chromosome of the bacterium. This DNA molecule was called Ti (tumor-inducing) plasmid (Zaenen et al., Chilton et al.).

The Ti plasmid contains most of the genes required for tumor formation. Wounded plants exude phenolic compounds that stimulate the expression of the virulence genes (vir -genes), which are also located on the Ti plasmid (Wullems et al. , Hoekema et al.). The vir genes encode a set of proteins responsible for the excision, transfer and integration of the T-DNA into the plant genome. The genes in the T-DNA region are responsible for the tumorigenic process. Some of them direct the production of plant growth hormones that cause proliferation of the transformed plant cells. The T-DNA region is flanked at both ends by 25 base pairs (bp) of nucleotides called T-DNA borders (Zambryski et al.). The T-DNA left border is not essential, but the right border is indispensable for T-DNA transfer.

The study of Agrobacterium and its natural mechanism to alter the biology of infected plant cells sparked the design of molecules that would transfer genes of interest into plant cells. These engineered DNA molecules are commonly referred to as vectors. The starting molecules can be native Ti-plasmids present in Agrobacterium or native or modified plasmids from other bacteria known to deliver DNA into transformed cells.

The basic elements of the vectors designed for Agrobacterium-mediated transformation that were taken from the native Ti-plasmid:

• the T-DNA border sequences, at least the right border, which initiates the integration of the T-DNA region into the plant genome
• the vir genes, which are required for transfer of the T-DNA region to the plant, and
• a modified T-DNA region of the Ti plasmid, in which the genes responsible for tumor formation are removed by genetic engineering and replaced by foreign genes of diverse origin, e.g., from plants, bacteria, virus. When these genes are removed, transformed plant tissues or cells regenerate into normal-appearing plants and, in most cases, fertile plants.

Although other methodologies for plant transformation have been devised, Agrobacterium remains one of the preferred mechanisms to introduce exogenous genes into the plant cells. One of the reasons for this is the wide spectrum of plants that are susceptible to transformation by this bacterium. Agrobacterium was initially believed to be restricted to the transformation of certain dicotyledonous plants (flowering plants with two cotyledons in their seeds and broad leaves) such as potato and tomato, but nowadays, transformation of monocotyledonous plants (flowering plants with one cotyledon in their seeds and narrow leaves with parallel veins), such as maize and rice is routinely performed.

Essential Features

Several essential features are required for Agrobacterium-mediated transformation of plants:

• vir genes. Approximately 35 vir genes map outside the T-DNA (transferred DNA) region and encode products required for excision, transfer, and integration of T-DNA into a plant genome. vir genes act in trans, meaning they do not need to be physically attached to the T-DNA to cause integration into the plant genome.
• T-DNA border sequences. These are sequences of 25 bp imperfect repeats that flank the T-DNA and are required for its transfer. Border sequences encompass the recognition sites for a site-specific endonuclease, which is encoded by the vir D operon, part of the vir genes. The endonuclease cleaves the lower DNA strand of the T-DNA marking the starting point of the transfer.

• Apart from the T-DNA border sequences, most of the genes of the T-DNA can be replaced by genes of interest to be transferred into the plant. Such engineered T-DNA is generally referred to as mutant T-DNA, engineered T-DNA or disarmed T-DNA. However, the exact definition of the terms in a patent application is often provided by the inventor.

• cis-regulatory regions. These include the right and left T-DNA borders, which are physically attached to the genes to be transferred into the plant genome. Other cis -regulatory regions include promoters and terminators that flank the transgenes and regulate their expression. Commonly used promoters and terminators are the nopaline synthesis gene (NOS) and Cauliflower Mosaic Virus (CaMV) 35S promoter.
• Selectable marker genes. In plants, they allow the identification and selection of cells with the gene of interest incorporated in their genome. Bacterial selectable markers permit the identification of bacteria transformed with a vector carrying the marker. Examples of plant and bacterial selectable markers are hygromycin phosphotransferase and kanamycin, respectively.

The above-mentioned elements are incorporated in two basic types of vectors used to transform a wide range of plants via Agrobacterium:

• Binary vectors. In this system, the T-DNA and the vir region reside in separate plasmids within the same Agrobacterium strain. The vir genes are located in a disarmed (without tumor genes) Ti plasmid and the T-DNA with the gene of interest is located in a small vector molecule.
• Co-integrated vectors. These vectors result from the recombination of a small vector plasmid, for example an E. coli vector, and a Ti plasmid harbored in A. tumefaciens. The recombination takes place through a homologous region present in both of the plasmids. An engineered T-DNA containing the gene of interest can be in either one of the plasmids.

References

Binary Vectors

The discovery that the vir genes do not need to be in the same plasmid with a T-DNA region to lead its transfer and insertion into the plant genome led to the construction of a system for plant transformation where the T-DNA region and the vir region are on separate plasmids.

In the binary vector system, the two different plasmids employed are:

• a wide-host-range small replicon, which has an origin of replication (ori) that permits the maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium. This plasmid typically contains:
  
  1. foreign DNA in place of T-DNA,
  2. the left and right T-DNA borders (or at least the right T-border),
  3. markers for selection and maintenance in both E. coli and A. tumefaciens,
  4. a selectable marker for plants.

• The plasmid is said to be "disarmed", since its tumor-inducing genes located in the T-DNA have been removed.

• a helper Ti plasmid, harbored in A. tumefaciens, which lacks the entire T-DNA region but contains an intact vir region.

In general, the transformation procedure is as follows:

• the recombinant small replicon is transferred via bacterial conjugation or direct transfer to
  A. tumefaciens harboring a helper Ti plasmid,
• the plant cells are co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNA into the plant genome, and
• transformed plant cells are selected under appropriate conditions.

  

  

Possible pitfalls

A possible disadvantage may ensue from the fact that the stability of wide host range replicons in E. coli and Agrobacterium varies considerably. Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli where both origins are active.

Advantages

Compared with co-integrated vectors, binary vectors present some advantages:

• No recombination process takes place between the molecules involved.
• Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium.

This vector system is most widely used nowadays. Different types of binary vectors have been devised to suit different needs in a plant transformation process.

Binary vector types

1. pGA series vectors, which contain:
   • an ori derived from RK2 for replication in E. coli and Agrobacterium,
   • a tetracycline resistance gene,
   • the cis-acting factor required for conjugal transfer,
   • the right (RB) and left (LB) T-DNA borders,
   • a neomycin phosphotransferase (nptII) gene, which confers resistance to kanamycin and G418 in transformed plants, and
   • a polylinker site (multicloning site).
   Specific vectors in this series are designed for cloning large fragments (colE1 origin of replication and phage l cos), analyzing promoters (multiple cloning site immediately upstream of a promoterless cat gene), and expressing a gene of interest (polylinker site between a plant promoter and a terminator).
2. pCG series vectors, which contain:
   • the origin of replication of the Agrobacterium rhizogenes root -inducing plasmid pRiHRI, which confers more stability in Agrobacterium than the ori derived from RK2, and a ColE1 origin of replication from the vector pBR322 for maintenance in E. coli.
3. pCIT series which contain:
   • the hygromycin (hph) resistance gene for plants,
   • the lambda cos site for cloning long fragments.
4. pGPTV (glucuronidase plant transformation vector) series , which have:
   • different plant selectable marker genes close to the left T-DNA border. This design overcomes problems inherent with the preferential right to left border transfer of T-DNA and improves the chances of having the gene of interest transferred to the plant cell in cells expressing the selectable marker gene.
5. pBECK2000 series, which contain:
   • synthetic T-DNA borders and a bar gene, which confers the plants resistance to the herbicide phosphinothricin. Also, the vectors use the phage P1 Cre/loxP site-specific recombinase system, which permits the transfer and integration of a target and marker genes as a single T-DNA unit into the plant genome or as two independent T-DNAs within a single Agrobacterium. It also allows site-specific excision of marker genes from the plant genome after transformation.
6. Binary-BAC (BiBAC) vector
   • based on a bacterial artificial chromosome (BAC) vector and is suitable for Agrobacterium-mediated transformation of high-molecular-weight DNA
   • comprises low-copy number origins of replication for both E. coli and Agrobacterium to ensure replication of the plasmid as a single-copy in both bacteria; and
   • a helper plasmid carrying additional copies of vir-genes in order to clone very large T-DNAs (up to 150 kb) into the plant genome.
7. pGreen series, small plasmids of around 3.2 Kb containing:
   • a broad host range replication origin (ori pSa) and a ColE1 origin derived from pUC,
   • a pSa replicase gene (rep A) that provides replication functions in trans and is located in a compatible plasmid (pSoup) in Agrobacterium, and
   • multiple cloning sites based on the pBlueScript vector, which allow any arrangement of selectable marker and reporter genes.

References

Co-integrated Vectors

Called co-integrated vectors or hybrid Ti plasmids, these vectors were among the first types of modified and engineered Ti plasmids devised for Agrobacterium -mediated transformation, but are not widely used today.

These vectors are constructed by homologous recombination of a bacterial plasmid with the T-DNA region of an endogenous Ti plasmid in Agrobacterium. Integration of the two plasmids requires a region of homology present in both.

Three vectors are necessary in this system:

• Disarmed Agrobacterium Ti plasmids
  In these Ti plasmids, the oncogenes located in the T-DNA region have been replaced by exogenous DNA.
  Examples of these vectors include:
  
  1. SEV series: the right border of the T-DNA together with the phytohormone genes coding for cytokinin and auxin are removed and replaced by a bacterial kanamycin resistance gene while the left border and a small part of the left segment (T_L) of the original T-DNA (referred to as Left Inside Homology (LIH)) are left intact.
  2. pGV series: the phytohormone genes are excised and substituted by part of pBR322 vector sequence. The left and right border sequences as well as the nopaline synthase gene of the Ti plasmid are conserved.

• Intermediate vectors
  These are small pBR322-based plasmids (E. coli vectors) containing a T-DNA region. They are used to overcome the problems derived from the large size of disarmed Ti plasmids and their lack of unique restriction sites. Intermediate vectors are replicated in E.coli and are transferred into Agrobacterium by conjugation. They cannot replicate in A. tumefaciens and therefore, carry DNA segments homologous to the disarmed T-DNA to permit recombination to form a co-integrated T-DNA structure.

• Helper vectors
  These are small plasmids maintained in E. coli that contain transfer (tra) and mobilization (mob) genes, which allow the transfer of the conjugation-deficient intermediate vectors into Agrobacterium.

A resulting co-integrated plasmid assembled by in vitro manipulation normally contains:

1. the vir genes,
2. the left and right T-DNA borders,
3. an exogenous DNA sequence between the two T-DNA borders, and
4. plant and bacterial selectable markers.

Some drawbacks

Although co-integrated vectors have been designed to allow site-specific recombination based on the recombination system of the phage P1 (e.g., wP1loxP-Cre series), co-integrated vectors in general are less popular due to:

• long homologies required between the Ti plasmid and the E. coli plasmids making them difficult to engineer and use, and
• relatively inefficient gene transfer compared to the binary vectors.

References

References

Essential features

Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids. Res. 12: 8711-8721.

Bomhoff, G., Klapwijk, P.M., Kester, H.C.M., and Schilperoort, R.A. 1976. Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet. 145: 177-181.

Braun, A.C.1958. A physiological basis for autonomous growth of the crown-gall tumor cell. Proc. Nat. Acad. Sci. USA 44: 344-349.

Braun, A.C. 1969. Abnormal growth in plants. In: Plant Physiology-A Treatise (Steward, F.C. Ed.) Acad. Press, N.Y., Vol VB: 379-420.

Chilton, M.D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: 263-271.

Chilton, M.D. 1983. A vector for introducing new genes into plants. Scientific American 248. 6: 36-45.

Drummond, M.H., Gordon, M.P., Nester, E.W., and Chilton, M.D. 1977. Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumors. Nature 269: 535-536.

Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, D., and Wu, R. 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnology 14: 494-498.

Grimsley, N., Hohn., T., Davies, J.W., and Hohn, B. 1987. Agrobacterium-mediated delivery of infectious maize streak virus into maize plants. Nature 325: 177-179.

Greene, E.A. and Zambryski, P.C. 1993. Agrobacteria mate in opine dens. Current Biology 3, 507-509. Herrera-Estrella, L., De Block, M., Messens, E., Hernalsteens, J.P., Van Montagu, M., and Schell, J. 1983a. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2: 987-995.

Herrera-Estrella, L., Depicker, A., Van Montagu, M., and Schell, J. 1983b. Expression of chimeric genes transferred into plant cells using a Ti plasmid-derived vector. Nature 303: 209-213.

Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179-180.

Hoekema, A., Hooykaas, P.J.J., and Schilperoort, R.A. 1984. Transfer or the octopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumor- or root-inducing plasmids: Generality of virulence systems. J. Bacteriol. 158: 383-385.

Hooykaas, P.J.J. and Schilperoort, R.A.1992. Agrobacterium and plant genetic engineering. Plant Mol. Biol. 19, 15-38. Hooykass-Van Slogteren, G.M.S., Hooykaas, P.J.J., and Schilperoort, R.A. 1984. Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens. Nature 311: 763-764.

Ishida, Y., Saitao, H., Ohta, S., Hiei, Y., Komari, T., and Kumashiro, T. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens . Nature Biotechnology 14: 745-750.

Ooms, G., Hooykaas, P.J.J., Moolenaar, G., and Schilperoort, R.A. 1981. Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions. Gene 14: 33-50.

Schell, J., and Van Montagu, M. 1983. The Ti plasmids as natural and as practical gene vectors for plants. Bio/Technology 1: 175-180.

Schilperoort, R.A., Veldstra, H., Warnaar, S.O., Mulder, G., and Cohen, J.A. 1967. Formation of complexes between DNA isolated from tobacco crown gall tumors and RNA complementary to Agrobacterium tumefaciens DNA. Biochem. Biophys. Acta 145: 523-525.

Smith, E.F. and Townsend, C.O. 1907. A plant tumor of bacterial origin. Science 25: 671-673.

Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort, R.A., and Schell, J. 1974. Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252: 169-170.

Van Larebeke, N., Genetello, C., Schell, J., Schilperoort, R.A., Hermans, A.K., Hernalstenns, J.P. and Van Montagu, M. 1975. Acquisition of tumor-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature 255: 742-743.

Willmitzer, L., De Beuckeleer, M., Lemmers, M., Van Montagu, M., and Schell, J. 1980. DNA from Ti plasmid present in nucleus and absent from plastids of crown gall plant cells. Nature 287: 359-361.

Wullems, G.J., Molendijk, L., Ooms, G., and Schilperoort, R.A. 1981a. Retention of tumor markers in F1 progeny plants from in vitro induced octopine and nopaline tumor tissues. Cell 24: 719-727.

Wullems, G.J., Molendijk, L., Ooms, G., and Schilperoort, R.A. 1981b. Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens induced transformation of cell regenerating protoplasts derived from Nicotiana tabacum. Proc. Natl. Acad. Sci. USA. 78: 4344-4348.

Yadav, N.S., Vanderleyden, J., Bennett, D.R., Barnes, W.M., and Chilton, M.D.1982. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc. Natl. Acad. Sci. USA 79: 6322-6326.

Zhu, J., Oger, P.M., Schrammeijer, B., Hooykaas, P.J., Farrand, S.K., and Winans, S.C. 2000. The bases of crown gall tumorigenesis. J. Bacteriol. 182 (14): 3885-3895.

Zupan, J., Muth, T.R., Draper, O.,and Zambryski, P. 2000. The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23: 11-28.

Binary Vectors

An, G. 1987. Binary Ti vectors for plant transformation and promoter analysis. Methods Enzymol. 153: 292-305.

An, G., Watson, B.D., Stachel, S., Gordon, M.P., and Nester, E.W. 1985. New cloning vehicles for transformation of higher plants. EMBO J. 4: 277-284.

An, G., Ebert, P.R., Mitra, A., and Ha, S.B. 1988. Binary vectors. In: Plant Molecular Biology Manual A3: 1-19. Gelvin., S.B., Schilperoort, R., and Verma, D. P. (Eds.), Kluwer Academic Pub., The Netherlands.

Becker, D., Kemper, E., Schell, J., and Masterson, R. 1992. New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol. Biol. 20: 1195-1197.

Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12: 8711-8721.

Fütterer, J. 1995. Expression signals and vectors. In: Gene Transfer to Plants, pp.311-324. Potrykus, I., Spangenberg, G. (Eds.), Springer-Verlag, Berlin.

Hamilton, C.M., Frary, A., Lewis, C., and Tanksley, S.D. 1996. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc. Natl. Acad. Sci. USA 93: 9975-9979.

Hamilton, C.M. 1997. A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene 200: 107-116.

Hellens, R., and Mullineaux, P. 2000. A guide to Agrobacterium binary Ti-vectors. Trends Plant Sci. 5: 446-451.

Hellens, R. et al. 2000. pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42: 819-832.

Hoekema, A., Hirsh, P.R., Hooykaas, P.J.J., and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179-180.

Ma, H., Yanofsky, M.F., Klee, H.J., Bowman, J.L., and Meyerowitz, E.M. 1992. Vectors for plant transformation and cosmid libraries. Gene 117: 161-167.

McBride, K.E. and Summerfelt, K.R. 1990. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 14: 269-276.

McCormac, A.C., Elliot, M.C., Chen, D.F. 1999. PBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates " clean-gene" facilities. Molecular and General Genetics 261: 226-235.

Co-integrated vectors

Christou, P. 1992. Genetic transformation of crop plants using microprojectile bombardment. Plant J. 2: 275-281.

Klee, H.J., F.F. White, V.N. Iyer, M.P. Gordon, and E.W. Nester. 1983. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J. Bacteriol. 153: 878-883.

Klee, H., Horsch, R., and Rogers, S. 1987. Agrobacterium-mediated plant transformation and its further applications to plant biology. Ann. Rev. Plant Physiol. 38: 467-486.

Klein, T.M., Wolf, E.D., Wu, R., and Sanford, J.C. 1987. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70-73.

Mozo, T., and Hooykaas, P.J.J. 1992. Design of a novel system for the construction of vectors for Agrobacterium -mediated plant transformation. Mol. Gen. Genet. 236: 1-7.

Neuhaus, G., and Spangenberg, G. 1990. Plant transformation by microinjection techniques. Physiol. Plant. 79: 213-217.

Ooms, G., P.M. Klapwijk, J.A. Poulis and R.A. Schilperoort. 1980. Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens J. Bacteriol. 144(1): 82-91.

Paszkowski, J., Saul, M.W., and Potrykus, I. 1989. Plant gene vectors and genetic transformation: DNA-mediated direct gene transfer to plants. In: Cell Culture and Somatic Cell Genetics of Plants (J. Schell and I.K. Vasil, Eds.). 6: 52-68.

Potrykus, I. 1995. Gene transfer to Plants. In: Gene transfer to Plants. I. Potrykus G. Spangenberg (Eds.). Springer-Verlag, Germany, pag III-IX.

Rogers, S.G., Klee, H.J., Horsch, R.B., and R.T. Fraley. 1987. Improved vectors for plant transformation: expression cassette vectors and new selectable markers. Methods in Enzymology. 153: 253-292.

Stachel, S.E., Timmerman, B., and Zambryski, P. 1986. Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to the plant cells. Nature 322: 706-712.

Stachel, S.E., and Zambryski, P. 1986. Vir A and virG control the plant induced activation of the T-DNA transfer process of Agrobacterium tumefaciens. Cell 46: 325-333.

Van Haute, L., Joos, H., Maes, M., Warren, G., Van Montagu, M., and Schell, J. 1983. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: A novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO Journal. 2: 411-417.

Yanofsky, M., Porter, S., Young, C., Albright, L., Gordon, M., and Nester, E. 1986. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47: 471-77.

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 callus, immature embryo, pollen, 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 transformation. Japan 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 recently abandoned. 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.

Callus Transformation - patents and application assigned to Japan Tobacco

Patents and application assigned to Japan Tobacco

In the disclosures, an explant of a monocot in the process of dedifferentiation or already dedifferentiated is used for transformation with Agrobacterium. A dedifferentiated tissue or a tissue in the process of dedifferentiation is described in the disclosures as an explant cultured on a dedifferentiation medium for not less than 7 days. Among the preferred tissues are a callus, an adventitious embryo-like tissue, and suspension cells.

Callus Transformation - patents granted to Japan Tobacco

Specific patent information

Note: Patent information on this page was last updated on 21 February 2006.

Immature embryo transformation - patents and application assigned to Japan Tobacco

Patents and application assigned to Japan Tobacco

This family of patents discloses use of an immature embryo of a monocot for Agrobacterium transformation. Within the embryo, the scutellum (name given to the single massive cotyledon (seed leaf) of monocot plants) is transformed. The scutellum is capable of producing dedifferentiated calli having the ability to regenerate normal plants after transformation.

The bacterium used for transformation contains either a Ti or Ri (root-inducing) plasmid with the desired gene and a plasmid having a virulence region derived from the A. tumefaciens Ti plasmid pTiBo542.

Immature embryo transformation - claims in plain English

Immature embryo transformation
Patent and application assigned to Japan Tobacco

Specific patent information

Note: Patent information on this page was last updated on 21 February 2006.

Pollen Transformation - patent granted to USDA

The invention is a method for the genetic transformation of any plant by using pollen as starting material for transformation with Agrobacterium. A culture medium useful for pollen germination and pollen tube growth in presence of Agrobacterium is also claimed.

Pollen Transformation - patent granted to USDA - claims in plain English

Pollen transformation
Patents granted to the United States Department of Agriculture (USDA)

Specific Patent Information

Note: Patent information on this page was last updated on 21 February 2006.

Patent application filed by the United States Department of Agriculture (USDA)

The invention disclosed in the European application is the same as in the United States patent and the recently granted Australian patent AU-B-733 080 (former Australian application AU 84005/98 A1).

Bibliographic data

Summary of the invention

The independent claims as filed in the EP application 996 328 A1 are similar to the granted claims of its related United States and Australian patents. As in the Australian patent, the EP application also contains a filed claim referring to a medium for pollen germination and pollen tube growth.

View Claims

EP Patent application filed by the United States Department of Agriculture (USDA)

Claims in plain English

Actual pending claims

Note: The Australian application AU 84005/98 A1 was granted. See Patents granted to the USDA for more information on this new patent.

* Identical to claim 1 of the granted United States and Australian patents.

**Identical to claim 8 of the granted Australian patent.

Shoot apex transformation - patent granted to The Texas A & M University System

In the invention disclosed in this United States patent, shoot apex tissue from any plant is subjected to gene transfer via Agrobacterium. According to the inventors, the use of such tissue permits rapid propagation of plants without encountering problems of somaclonal variation.

Shoot apex transformation - - claims in plain english - patent granted to The Texas A & M University System

Specific Patent Information

Note: Patent information on this page was last updated on 22 February 2006.

In planta transformation

Overview

The following patents are directed to the transformation of a plant in vivo with Agrobacterium, in which the inoculation and co-cultivation process with Agrobacterium takes place as the plant develops normally.

As described in some of the patents, some advantages of this methodology derive from a close analogy to Agrobacterium's natural environment for transformation and the production of non-chimeric transgenic progeny from seeds of a treated plant when floral tissue is transformed.

Cotton Inc., Paradigm Genetics Inc , and Rhobio patents and applications are presented here.

Cotton Inc. claims

• the introduction of Agrobacterium into the floral tissue of a plant using a needleless-injection device. Development of transformed seed takes place in the plant. More information about this invention

Paradigm Genetics Inc claims (Update July 2003)

• a method for preparing a transformed plant or seed in which the Agrobacterium carrying the DNA to be transformed is grown to a certain density, diluted in an aqueous medium and then applied to floral tissues for transformation. More information about this invention