Patent applications filed by The Samuel Roberts Noble Foundation

The applications describe methods for direct plant transformation via Agrobacterium using vacuum. In one of the disclosures, Agrobacterium containing a vector with a gene of interest contacts the aerial portions of a plant at flowering stage under vacuum conditions. The vacuum applied is of sufficient strength to force the Agrobacterium cells into intimate contact with the plant such that the T-DNA transfer to the plant takes place.

A plant at flowering stage is defined as a plant form about the beginning of the first flower bud formation to about the time of the last flower set. The flowering plants are grown from vernalised seeds. These are seeds subjected to a period of chilling before germination. The seeds are incubated at 4°C for a period of time, preferably in the dark, and kept moist. Vernalisation is used to speed up the formation of flowers.

In the other disclosure, the material selected for transformation is a seedling.

As part of both disclosures, the plant material is transformed with a mix of Agrobacterium cells containing different T-DNA to be inserted into the plants. The transformed plant is allowed to grow into maturity and produce seeds. Progeny from the seed is selected by the use of selectable markers and the presence of an additional transferred gene.

Specific PCT Application Information

PCT Application Number Title, Independent Claims and Summary of Claims Assignee
WO 2000/037663 A2

  • Earliest priority – 23 December 1998
  • Filed – 23 December 1999
  • OPI – 29 June 2000
  • Expected expiry – N/A
Title – Plant transformation process

Claim 1
A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling saidAgrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different.

Claim 9A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different.

Claim 18A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling saidAgrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force saidAgrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different;
C) allowing said transformed seedling to grow to maturity and set seed;
D) germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression;
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

The claims as filed of the PCT application WO 2000/037663 recite:

  • a method for direct transformation of seedlings with Agrobacterium by placing the plant material with Agrobacterium having a vector with a gene of interest or a gene fragment in its T-DNA and applying vacuum to them so Agrobacterium enters into contact with the seedling and transfers the T-DNA to the plant cells;
  • a method as the one described above but the material is in contact with a mixture of Agrobacterium cells harboring different DNA fragments (aren’t necessarily gene or gene fragments) in their T-DNAs; and
  • a method as the one first mentioned where after the transformation plants set seed and the progeny from these seeds is selected for the presence of a gene of interest and a selectable marker.

Status of National phase entries listed on INPADOC:

  1. Australia (AU25943/00 A) – application lapsed on 28 July 2005
  2. Canada (CA2352488 A1) – dead application on 23 December 2004 (i.e., the application cannot be reinstated).
  3. Europe (EP1141356 A2) – application deemed to be withdrawn on 2 February 2005
  4. Japan (JP2002533090 T) – application pending
The Samuel Roberts Noble Foundation
WO 2000/063400 A2

  • Earliest priority – 21 April 1999
  • Filed – 20 April 2000
  • OPI – 26 October 2000
  • Expected expiry – N/A
Title – Plant transformation process

Claim 1A method for direct plant transformation using plants and Agrobacterium comprising:

A) contacting the aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

Claim 9A method for direct transformation of a plant comprising:

A) vernalizing and germinating initial seed to form said plant contacting the aerial portions of said plant at the time of flowering withAgrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

Claim 17A method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising:
A) contacting aerial portions of at least one plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.
Claim 25A method for direct transformation of a plant at the time of flowering comprising:

A) vernalizing and germinating initial seed to form said plant contacting aerial portions of said plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

Claim 33
A method for direct plant transformation using plants at the time of flowering and Agrobacterium

comprising:A) contacting aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
C) allowing said transformed plant to grow to maturity and set seed;
D) germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

Claim 36A method for direct transformation of a plant at the time of flowering comprising:

A) vernalizing and germinating initial seed to form said plant;
B) contacting aerial portions of said plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
C) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
D) allowing said transformed plant to grow to maturity and set seed; germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

Status of National phase entries listed on INPADOC:

  1. Australia (AU43652/00 A) – application lapsed on 8 December 2005
  2. Canada (CA2370638 A1) – dead application on 20 April 2005 (i.e., the application cannot be reinstated).
  3. Europe (EP1171618 A2) – application deemed to be withdrawn on 11 May 2005
  4. New Zealand (NZ513993 A) – application void on 30 January 2004.

Note: Patent information on this page was last updated on 8 March 2006.