Inhibition of Agrobacterium growth

PCT and US application filed by Monsanto

Overgrowth of Agrobacterium jeopardizes the survival of the transformed plant cells and also has an effect on the T-DNA transfer process. Insertion of multiple copies of a gene of interest into a plant cell is influenced by the frequency of T-DNA transfer into the cell. Agrobacterium-mediated transformation protocols strive to attain transformation events with a limited number of copies of DNA entering into any one cell. The presence of multiple inserts can lead to gene silencing or reduce expression levels of transformed genes, which is caused by several mechanisms including recombination between the multiple copies. Inhibiting agents of Agrobacterium growth should be effective against the bacterium but remain neutral with respect to plant cell growth.

Monsanto’s applications relate to the control of Agrobacterium growth during the transformation process in order to improve transformation efficiency. The use of inhibiting agents during inoculation and co-culture of Agrobacterium with a transformable plant cell results, according to the disclosure, in increased transformation efficiencies and a low copy number of an introduced genetic component in several plant systems. Preferred growth-inhibiting agents are compounds containing heavy metals such as silver nitrate or silver thiosulfate, antibiotics such as carbenicillin, and a combination of antibiotics and a clavulanic acid such as augmentin or timentin.

PCT Application Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
WO 2001/09302

  • Earliest priority – 29 July 1999
  • Filed – 28 July 2000
  • OPI – 8 February 2001
  • Expected Expiry – N/A
Title – A novel Agrobacterium-mediated plant transformation method

Claim 1

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:
A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media not containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed plant cells or tissue.

Claim 23A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of: A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.
Claim 45A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:
A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of the plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

The claims as filed of the PCT application recite:

  • methods of transforming plant cells or tissues with Agrobacterium , where the growth of Agrobacterium cells is inhibited during:
    • the inoculation phase, where Agrobacterium and plant cells are first brought into contact with each other;
    • the co-cultivation phase, where Agrobacterium and plant cells are grown together for for an unspecified period; or
    • both the inoculation and co-cultivation phases.
Monsanto
US 2003204875 AA

  • Earliest priority – 29 July 1999
  • Filed – 17 April 2003
  • Published -30 Oct 2003
  • Status -pending
Title – Novel Agrobacterium-mediated plant transformation method

Claim 1

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

  • inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
  • co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media not containing a growth inhibiting agent;
  • selecting transformed plant cells or tissue; and
  • regenerating a transformed plant expressing the genetic component from the selected transformed plant cells or tissue.
Claim 23
A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

  • inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue;
  • co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
  • selecting transformed plant cells or tissue; and
  • regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.
Claim 45
A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

  • inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
  • co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of the plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
  • selecting transformed plant cells or tissue; and
  • regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

This application is a division of  US 6603061, which is the priority document of WO 2001/09302.  Claims of granted US 6603061 recite a method of Agrobacterium-mediated transformation that is limited to that transforming a corn plant (claim 1) or a dicotyledonous plant (claim 4).

Remarks
  1. National phase entry of WO 2001/09302 in Australia (AU 200063892) has lapsed on 3 April 2003.
  2. National phase entry of WO 2001/09302 in Canada (CA 2381254) is deemed dead on 28 July 2004.
  3. National phase entry of WO 2001/09302 in Europe (EP 1200613) and Japan (JP 2003506035) are pending.
  4. Other national phase entry of WO 2001/09302 includes: Brazil (BR 200013187).

Note: Patent information on this page was last updated on 6 March 2006.