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Patent granted to Washington University

The present United States patent granted to Washington University discloses the transformation of dicot plants with an Agrobacterium vector having the cytokinin gene of the T-DNA region inactivated.  According to the USPTO assignments database, this patent was exclusively licensed to Syngenta.

In a wildtype T-DNA of a Ti plasmid, the genes encoding phytohormones are responsible for the tumorous state of a transformed tissue. Cytokinin, one of those phytohormones, induces the formation of shoots in a tumor.

In the disclosed invention, regeneration of a transformed dicot plant is achieved by inactivating the cytokinin gene. Additionally, the cytokinin gene is replaced by foreign DNA. The plasmid containing the mutant T-DNA, with foreign DNA replacing the cytokinin gene, is accomplished by homologous recombination within Agrobacterium.

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6051757

  • Earliest priority – 4 November 1983
  • Filed – 5 June 1995
  • Granted – 18 April 2000
  • Expected expiry – 17 April 2017
 Title – Regeneration of plants containing genetically engineered T-DNA

Claim 1A method of transforming a dicotyledonous plant susceptible to transformation by Agrobacterium, comprising:

contacting the plant with an Agrobacterium tumefaciens bacterium comprising a gene vector, the vector comprising
(i) DNA foreign to the Agrobacterium, and
(ii) the vector not comprising a functional cytokinin autonomy gene.
Claim 2A method for producing a morphologically and developmentally normal dicotyledonous plant comprising non-Agrobacteriumforeign DNA stably integrated in the plant’s genome, said method comprising the following steps:

A) transforming a dicotyledonous plant cell susceptible to transformation by Agrobacterium with an Agrobacterium -derived gene vector, said vector comprising
(i) non-Agrobacterium foreign DNA and
(ii) the vector not comprising a functional cytokinin autonomy gene; and

B) regenerating said transformed plant cell to produce a morphologically and developmentally normal transformed plant with said foreign DNA stably integrated in the plant’s genome.
Claim 5A method for producing a transgenic dicotyledonous plant comprising a stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) sexually propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said foreign DNA having been introduced into the genome by an Agrobacterium-derived gene vector not comprising a functional cytokinin-autonomy gene; and
B) selecting for progeny plants which comprise the non-Agrobacterium foreign DNA stably integrated into the genome of said progeny plants.
Claim 6A method for producing a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) sexually propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) obtaining a progeny plant which comprises the non-Agrobacterium foreign DNA stably integrated into its genome.
Claim 7A method for producing a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) obtaining a plant which comprises the non-Agrobacterium foreign DNA stably integrated into its genome.
Claim 8A method for producing a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

growing a seed of a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene.
Claim 9A method for producing seed of a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) harvesting seed from said propagated plant.
Claim 10A method of transforming a dicotyledonous plant of a species that is a naturally susceptible host for Agrobacterium, comprising:

contacting the plant with an Agrobacterium bacterium comprising a gene vector, the vector comprising
(i) DNA foreign to the Agrobacterium and
(ii) the vector not comprising a functional cytokinin autonomy gene.
Claim 11A method for producing a transgenic dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated in the plant’s genome, said method comprising the following steps:

A) transforming a cell of a dicotyledonous plant species that is a naturally susceptible host for Agrobacterium by Agrobacterium -mediated transformation with a gene vector comprising
(i) non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) regenerating said transformed plant cell to produce a normal transformed dicotyledonous plant with said foreign DNA stably integrated in the plant’s genome.
Claim 14A method for producing a transgenic dicotyledonous plant comprising a stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) sexually propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which is of a species that is a naturally susceptible host for Agrobacterium and which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) obtaining a progeny plant which comprises the non-Agrobacterium foreign DNA stably integrated into its genome.
Claim 15A method for producing a transgenic dicotyledonous plant comprising a stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which is of a species that is a naturally susceptible host for Agrobacterium and which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) obtaining a plant which comprises the non-Agrobacterium foreign DNA stably integrated into its genome.
Claim 16A method for producing a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

growing a seed of a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which is of a species that is a naturally susceptible host for Agrobacterium and which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene.
Claim 17A method for producing seed of a transgenic dicotyledonous plant comprising stably integrated non-Agrobacterium foreign DNA, the method comprising:

A) propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which is of a species that is a naturally susceptible host for Agrobacterium and which was transformed by Agrobacterium-mediated transformation with a gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) harvesting seed from said propagated plant.
Claim 18An Agrobacterium-mediated method for genetically engineering a dicotyledonous plant comprising:

A) producing a transgenic plant cell by transforming a cell of dicotyledonous plant species that is a naturally susceptible host for Agrobacterium with a gene vector comprising non-Agrobacterium foreign DNA and not comprising a functional cytokinin autonomy gene by Agrobacterium tumefaciens-mediated transformation; and
B) regenerating a whole normal plant from the transgenic plant cell which contains said foreign DNA stably integrated into its genome.
Claim 20A method for producing a transgenic dicotyledonous plant comprising intact T-DNA comprising non-Agrobacterium foreign DNA stably integrated into the genome of said plant, the method comprising:

A) propagating a dicotyledonous plant comprising non-Agrobacterium foreign DNA stably integrated into its genome, said plant derived from a dicotyledonous plant which is of a species that is a naturally susceptible host for Agrobacterium and which was transformed by Agrobacterium-mediated transformation with a disarmed T-DNA gene vector comprising
(i) said non-Agrobacterium foreign DNA and
(ii) not comprising a functional cytokinin autonomy gene; and

B) obtaining a plant which comprises the intact T-DNA stably integrated into its genome.

The United States patent US 6051757 claims

  • transformation of a dicot plant, which is either susceptible to transformation (claim 1) or a naturally susceptible host (claim 10) with A. tumefaciens (claim 1) or an Agrobacterium (claim 10) having a vector that lacks the cytokinin function and contains foreign DNA instead;
  • regeneration of a transformed dicot cell and production of a morphologically normal plant;
  • sexual or non-sexual propagation of the transformed dicot plant and generation of a progeny bearing the foreign gene;
  • seed production and harvesting from a propagated transformed dicot plant.

Washington University, exclusively licensed toSyngenta
Remarks A related United States application (US 07/155092) was in interference, the process by which the United States Patent Office determines who was the earliest inventor when there are competing claims (in this case, from Monsanto).   According to the USPTO status database PAIR, Syngenta lost the interference case in 2004.

Note: Patent information on this page was last updated on 10 March 2006.