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Granted patent and applications filed by Syngenta (formerly Novartis)

According to the applicants the necrosis seen in some plants, i.e. Gramineae, upon Agrobacterium exposure is a programmed cell death that is different from the passive death experienced during oxidative browning and exposure to toxins. It is an active process in which the cells undergo morphological changes in part as a result of de novo gene expression and DNA cleavage.

The patents and patent applications disclose the use of physical and chemical methods for inhibiting Agrobacterium-induced necrosis (AIN). Heat shock treatment is one of the physical methods and among the chemical methods;chemical compounds are used as inhibiting agents of AIN. Nucleotide sequences such as p35 and iap (see below) stably or transiently expressed in the cell to be transformed also inhibit AIN. In addition, the applicants teach the use of Agrobacterium strains that are less likely to induce necrosis in the transformed tissue.


Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6162965

  • Earliest priority – 2 June 1997
  • Filed – 2 June 1998
  • Granted – 19 December 2000
  • Expected expiry – 1 June 2018
 Title – Plant transformation methods

Claim 1
A method for transforming a plant cell or tissue with a gene construct, comprising heat shocking said plant cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 2
A method for producing a fertile transgenic plant comprising a gene construct, which method comprises: (a) transforming a plant cell or tissue comprising heat shocking said plant cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said plant cell or tissue and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic plant.
Claim 12
A method for transforming a maize cell or tissue with a gene construct, comprising heat shocking said maize cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 13
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising heat shocking said maize cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.
Claim 14
A method for transforming a plant cell or tissue with a gene construct, comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said plant cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 15
A method for producing a fertile transgenic plant comprising a gene construct, which method comprises: (a) transforming a plant cell or tissue comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said plant cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic plant.
Claim 24
A method for transforming a maize cell or tissue with a gene construct, comprising exposing said maize cell or tissue to Agrobacteriumunder conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said maize cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 25
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said maize cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.
Claim 26
A method for transforming a gramineceous plant cell or tissue with a gene construct, comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said plant cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 27
A method for producing a fertile transgenic gramineceous plant comprising, a gene construct, which method comprises: (a) transforming a gramineceous plant cell or tissue comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibitAgrobacterium induced necrosis, wherein said conditions comprise culturing said plant cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and saidAgrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic gramineceous plant.
Claim 33
A method for transforming a maize cell or tissue with a gene construct, comprising exposing said maize cell or tissue to Agrobacteriumunder conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said maize cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.
Claim 34
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said maize cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.
Claim 35
A transgenic plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which comprises a coding sequence of a p35, iap or dad-1 gag.
Claim 36
A transgenic plant, plant tissue or plant cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of a p35, iap or dad-1 gene.
Claim 40
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin comprising a coding sequence of a p35 gene.
Claim 41
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of an iap gene.
Claim 42
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of a dad-1 gene.
Claim 44
A gramineceous plant cell or tissue culture medium, comprising (a) (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor; and (b) an Agrobacterium comprising a plasmid comprising a gene construct.

United States patent US 6162965 claims several methods to inhibit AIN in plants:

  • heat shock to treat plant cells or tissues before co-cultivating with Agrobacterium
  • transformation of a plant cell via Agrobacterium with sequences such as:
    • p35 and iap, which are apoptosis-inhibiting genes from baculovirus; and
    • dad-1, a gene capable of suppressing disease response in plants.

The patent also claims methods for inhibiting AIN in Gramineae in general and in maize in particular. Besides the methods mentioned above, gramineaceous plants and maize may be cultured in a necrosis inhibiting medium containing an inhibitor of ethylene or ethylene biosynthesis.

 Syngenta
US 2002/88029 AA

  • Earliest priority – 2 June 1997
  • Filed – 19 December 2000
  • Publication date – 4 Jul 2002
  • Expected Expiry – N/A
  • Status -Application abandoned 25 July 2005
 Title – Plant transformation methods

Claim 1

A method of transforming a plant cell with a gene of interest, comprising

  • exposing said plant cell to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN), wherein saidAgrobacterium comprises a vector comprising said gene of interest.
Claim 8
A method of making a fertile, transgenic plant comprising

  • transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN) and regenerating tissue thus transformed, wherein said Agrobacterium comprises a vector comprising a gene of interest.
Claim 9
A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.
Claim 10
A plant cell or tissue culture medium, comprisinga) a chemical inhibitor of AIN,
b) an Agrobacterium comprising a plasmid comprising a gene of interest, and
c) water and essential salts.
Claim 11
A method of transforming a totipotent cell of a plant of the family Gramineae, comprising exposing a population of said totipotent cells toAgrobacterium comprising a plasmid comprising a gene of interest, wherein the Agrobacterium is of a strain which does not induce significant levels of necrosis in said population at an exposure duration and concentration sufficient to achieve transformation of said cell.
Claim 12
A method for determining the suitability of an Agrobacterium strain for use in the transformation of a regenerable cell of a plant of the family Gramineae comprising exposing a population of said regenerable cells of the plant to the Agrobacterium strain and observing the necrosis in said cell population.
Claim 13
An Agrobacterium strain which has been genetically modified to reduce or eliminate expression of the Agrobacterium necrosis factor or a derivative of such a modified strain.

United States application US 2002/88029 is a continuation of application No. 09/089,111, which is now patent US 6162965 described above. The additional elements in this application include a method to select an Agrobacterium strain with reduced necrosis-inducing capacity from a population of regenerable plant cells from the family Gramineae. Further, the application also claims the Agrobacterium strain with reduced or eliminated production of the necrosis factor, obtained through the selection process or through genetic manipulation. Finally, a method is claimed to transform regenerable cells from the family Gramineae using such strains.

The “Agrobacterium necrosis factor” is  disclosed as “the heat labile factor observed in concentrated supernatant capable of inducing necrosis, e.g., programmed cell death, in maize embryos.” The application discloses sequences cloned into three BAC vectors associated with affects on cell death.  The sequences are reported to have homology with virB1, xylA-xylB and acvB respectively.  It’s not clear if the Agrobacterium strain referred to in claim 13 is required to reduce or eliminate the expression of one or all of these sequences since none of these sequences are clearly identified as “the” factor.  The genetic structure of a strain having the properties recited in the claims is not disclosed nor is a way of identifying such a strain disclosed.
AU 735472 B

  • Earliest priority – 2 June 1997
  • Filed – 29 May 1998
  • Granted – 12 July 2001
  • Expected expiry – 28 May 2018
 Title – Plant transformation methods

Claim 1

A method of transforming a plant cell or tissue with a gene of interest, comprising:

  • exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium-induced necrosis (AIN), wherein
    said Agrobacterium comprises a vector comprising said gene of interest, wherein
    said conditions which inhibit AIN comprise:

    • exposing said plant cell or tissue to Agrobacterium after heat shock treatment; or
    • exposing said plant cell or tissue to Agrobacterium in the presence of an agent inhibiting AIN, wherein said agent comprises:
      • a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors; or
      • a nucleotide sequence encoding mRNA or protein inhibiting AIN.
Claim 18A method of making a fertile, transgenic plant comprising: A) transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium -induced necrosis (AIN); and
B) regenerating tissue thus transformed, wherein
said Agrobacterium comprises a vector comprising said gene of interest, wherein
said conditions which inhibit AIN comprise:

  • exposing said plant cell or tissue to Agrobacterium after heat shock treatment; or
  • exposing said plant cell or tissue to Agrobacterium in the presence of an agent inhibiting AIN, wherein said agent comprises:
    • a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors; or
    • a nucleotide sequence encoding mRNA or protein inhibiting AIN.
Claim 39A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.
Claim 50A plant cell or tissue culture medium, comprising:

  1. a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors;
  2. an Agrobacterium comprising a plasmid comprising a gene of interest; and
  3. water and essentials salts.

The methods for inhibiting AIN claimed in the granted Australian patent are similar to the ones claimed in the United States patent. However, the group of chemical inhibitors additionally includes gibberellin antagonists and phosphatase inhibitors. Also, the nucleotide sequences whose products inhibit AIN are not limited to specific genes, but encode any mRNA or protein inhibiting AIN. Furthermore, unlike the United States patent, the Australian patent does not claim transformation of Gramineae or maize in particular.
WO 1998/54961 A2

  • Earliest priority – 2 June 1997
  • Filed – 29 May 1998
  • Publication date – 10 December 1998
 Title – Plant transformation methods

Claim 1

A method of transforming a plant cell with a gene of interest, comprising

  • exposing said plant cell to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN), wherein saidAgrobacterium comprises a vector comprising said gene of interest.
Claim 8
A method of making a fertile, transgenic plant comprising

  • transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN) and regenerating tissue thus transformed, wherein said Agrobacterium comprises a vector comprising a gene of interest.
Claim 9
A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.
Claim 10
A plant cell or tissue culture medium, comprisinga) a chemical inhibitor of AIN,
b) an Agrobacterium comprising a plasmid comprising a gene of interest, and
c) water and essential salts.
Claim 11
A method of transforming a totipotent cell of a plant of the family Gramineae, comprising exposing a population of said totipotent cells toAgrobacterium comprising a plasmid comprising a gene of interest, wherein the Agrobacterium is of a strain which does not induce significant levels of necrosis in said population at an exposure duration and concentration sufficient to achieve transformation of said cell.
Claim 12
A method for determining the suitability of an Agrobacterium strain for use in the transformation of a regenerable cell of a plant of the family Gramineae comprising exposing a population of said regenerable cells of the plant to the Agrobacterium strain and observing the necrosis in said cell population.
Claim 13
An Agrobacterium strain which has been genetically modified to reduce or eliminate expression of the Agrobacterium necrosis factor or a derivative of such a modified strain.

The claims as filed in the PCT application are broader in scope than the granted claims of both the Australian and the US patents. For example, one claim broadly recites “conditions which inhibit AIN” without specifying details of suitable conditions. In another claim the culture medium for the plant cell or tissue contains a chemical inhibitor without defining what sort of chemical. A couple of claims of the European application make reference to the use of an Agrobacterium strain that does not induce significant levels of necrosis. The Agrobacterium strain has been modified to reduce or eliminate expression of a necrosis factor. This strain of Agrobacterium may be used for transforming a totipotent Gramineae cell.
Remarks
  1. National phase entries of WO 1998/54961 in Canada (CA 2290863), Europe (EP 986299) and Japan (JP 2002502252) are pending.
  2. National phase entry of WO 1998/54961 in China (CN 1150320) has been granted on 19 May 2004.
  3. Other national phase entries listed in INPADOC include: Hungary (HU 200002903), Israel (IL 132768), Poland (PL 336979), Russian Federation (RU 2226549), Turkey (TR 200402566), Ukraine (UA 72443), South Africa (ZA 9804681).

Note: Patent information on this page was last updated on 3 March 2006.