Process for the incorporation of foreign DNA into the genome of dicotyledonous plants

• US 5,464,763 A
• Filed: 12/23/1993
• Issued: 11/07/1995
• Est. Priority Date: 02/24/1983
• Status: Expired due to Term

• Current Assignee: Syngenta Mogen BV (China National Chemical Corporation)
• Original Assignee: Rijksuniversiteit te Leiden, Schilperoort; Robbert Adriaan
• Inventors: Hoekema, Andreas, Schilperoort, Robbert A.
• Primary Examiner(s): MOODY, PATRICIA
• Application Number:
  US08/173,271
• Time in Patent Office: 684 Days
• Field of Search: 435/172.3, 435/240.4, 435/320.1, 435/252.3, 800/205, 935/30, 935/56, 935/67
• US Class Current: 800/294
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation

Abstract

The invention relates to a process for the incorporation of foreign DNA into chromosomes of dicotyledonous plants by infecting the plants or incubating plant protoplasts with Agrobacterium bacteria, which contain one or more plasmids, wherein bacteria are used which contain at least one plasmid having the vir-region of Ti (tumor inducing) plasmid but no T-region, and at least one other plasmid having a T-region with incorporated therein foreign DNA but no vir-region, as well as to a Agrobacterium bacteria, suitable for use in the process according to claim 1 wherein at least one plasmid which has the vir-region of a Ti (tumor inducing) plasmid but no T-region and at least one other plasmid which has a wild type T-region with incorporated in it foreign DNA but no vir-region.

Claims

1. A process for incorporating into the genome of dicotyledonous plants foreign DNA, comprising infecting the plants or plant cells or incubating plant protoplasts with Agrobacterium strains, which contain plasmids, said Agrobacterium strains containing at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said border sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

4. A process according to claim 1, wherein said foreign DNA comprises a gene including a plant promoter as starting place for the transcription of said gene in RNA.

5. A process according to claim 4, wherein said foreign DNA comprises in addition to said gene a marker gene for the selection of transformants, including a plant promoter as a starting place for the transcription of said marker gene in RNA.

6. A process according to claim 1, wherein the plasmid having a T-region but no vir-region is stable in said Agrobacterium strain.

7. A process according to claim 1, wherein the plasmid having a T-region but no vir-region, and the plasmid having a vir-region but no T-region do not form cointegrates at a level detectable by a bacterial conjugation test.

8. A process according to claim 1, wherein cointegration of said T-region plasmid and said vir-region plasmid do not contribute detectably to the said process.

9. A process according to claim 1, wherein frequency of cointegrate formation between said T-region plasmid and said vir-region plasmid is less than 1:

10,000.

10. A process according to claim 1, wherein said vir-region plasmid is pAL4404, as present in LBA4404, deposited under No. CBS191.83.

11. A process according to claim 1, wherein said T-region plasmid has only one right T-DNA border, and only one left T-DNA border.

2. Agrobacterium strains, comprising at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

3. A process for the production of Agrobacterium strains comprising at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said border sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation, said process comprising:

(a) incorporating non-Agrobacterium foreign DNA into a plasmid having a T-region and a replicator having a broad bacterial host range, wherein said foreign DNA becomes part of the T-region,

(b) cloning the resulting plasmid in Escherichia coli; and

(c) introducing the resulting plasmid into Agrobacterium strains which contain at least one plasmid which has the vir-region of a Ti-plasmid but no T-region.

12. A process for incorporating in the genome of dicotyledonous plants foreign DNA, comprising infecting the plants or plant cells or incubating plant protoplasts with Agrobacterium strains, which contain plasmids, said Agrobacterium strains containing at least one plasmid having the vir-region of a Ti-plasmid but no T-region, and at least one other plasmid having a single T-region but no vir-region, said single T-region being composed of naturally occurring border sequences at the extremities of said single T-region, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.