Claims
- 1. A method for targeting the insertion of a nucleotide sequence of interest into a target site within the genome of an acceptor plant, said method comprising:
a) sexually crossing a donor plant and the acceptor plant to produce a hybrid cell wherein,
i) the genome of the donor plant comprises at least one DNA construct comprising a transfer cassette comprising in series, a first recombination site, said nucleotide sequence of interest, and a second recombination site, wherein the first and second recombination sites are non-identical; ii) the genome of said acceptor plant comprises the target site, said target site comprising in series, the first recombination site, a DNA sequence, and the second recombination site, wherein the first and the second recombination sites are non-identical and correspond to the non-identical sites of the transfer cassette; and, iii) the acceptor plant and the donor plant are from different species; b) providing a recombinase, or variant or fragment thereof, that implements recombination at the non-identical recombination sites such that the nucleotide sequence of interest is inserted at the target site; c) recovering a transgenic haploid cell comprising the nucleotide sequence of interest, wherein the transgenic haploid cell is capable of generating a plant; and, d) generating a haploid transgenic plant comprising the nucleotide sequence of interest integrated into the target site.
- 2. The method of claim 1, further comprising generating a diploid transgenic plant comprising the nucleotide sequence of interest.
- 3. The method of claim 1, wherein the donor plant and the acceptor plant are from the same family.
- 4. The method of claim 3, wherein the donor plant and the acceptor plant are from the family Poaceae.
- 5. The method of claim 4, wherein the donor plant and the acceptor plant are from different genera, said genera selected from the group consisting of Zea, Triticum, Hordeum, Sorghum, Oryza, and Avena.
- 6. The method of claim 5, wherein the donor plant is from the genus Zea and acceptor plant is from the genus Triticum.
- 7. The method of claim 1, wherein the donor plant and the acceptor plant are from the same genus.
- 8. The method of claim 7, wherein the genus is selected from the group consisting of Zea, Triticum, Hordeum, Sorghum, Oryza, and Avena.
- 9. The method of claim 1, wherein said non-identical recombination sites are selected from the group consisting of FRT, mutant FRT, LOX and mutant LOX sites.
- 10. The method of claim 9, wherein the recombinase is selected from the group consisting of FLP, Cre, and chimeric FLP/Cre.
- 11. The method of claim 9, wherein said sites are selected from the group consisting of FRT and mutant FRT sites.
- 12. The method of claim 11, wherein said mutant FRT sites are selected from the group consisting of FRT5, FRT6 and FRT7.
- 13. The method of claim 11, wherein said recombinase is FLP, or an active variant or fragment thereof.
- 14. The method of claim 1, wherein a nucleotide sequence encoding said recombinase is stably incorporated into the genome of said acceptor plant, and said nucleotide sequence encoding said recombinase is operably linked to a promoter.
- 15. The method of claim 1, wherein said target site further comprises a nucleotide sequence encoding said recombinase, said nucleotide sequence encoding said recombinase is operably linked to a promoter, and wherein the nucleotide sequence encoding the recombinase is located between the first and the second recombination sites.
- 16. The method of claim 1, wherein the target site further comprises a selectable marker located between the first and the second recombination sites.
- 17. The method of claim 1, wherein the DNA construct comprising the transfer cassette further comprises a selectable marker located between the first and the second recombination sites.
- 18. The method of claim 17, wherein the selectable marker is not operably linked to a promoter, and wherein the target site further comprises a promoter active in said plant, such that the promoter is operably linked to the first recombination site of the target site, whereby insertion of the selectable marker at the target site results in the selectable marker being operably linked to the promoter.
- 19. A method for the stable introduction of a nucleotide sequence of interest into the genome of a plant, said method comprising:
a) sexually crossing a donor plant and an acceptor plant to produce a hybrid cell wherein,
i) the genome of said donor plant comprises at least one DNA construct comprising a transfer cassette comprising in series, a first recombination site, an expression cassette comprising said nucleotide sequence of interest, and a second recombination site, wherein the first and the second recombination sites are non-identical, ii) the genome of said acceptor plant comprises a target site comprising in series, the first recombination site, a DNA sequence, and the second recombination site, wherein the first and the second recombination sites are non-identical and correspond to the non-identical sites of the transfer cassette; and, iii) the acceptor plant and the donor plant are from different species; b) providing a recombinase or variant or fragment thereof, that implements recombination at the non-identical recombination sites such that the nucleotide sequence of interest is inserted at the target site; c) recovering a transgenic haploid cell comprising the nucleotide sequence of interest, wherein said haploid cell is capable of generating a plant; and, d) generating a haploid transgenic plant comprising the nucleotide sequence of interest is stably incorporated into its genome.
- 20. The method of claim 19, further comprising generating a diploid transgenic plant comprising the nucleotide sequence of interest.
- 21. The method of claim 19, wherein the donor plant and the acceptor plant are from the same family.
- 22. The method of claim 21, wherein the donor plant and the acceptor plant are from the family Poaceae.
- 23. The method of claim 22, wherein the donor plant and the acceptor plant are from different genera, said genera selected from the group consisting of Zea, Triticum, Hordeum, Sorghum, Oryza, and Avena.
- 24. The method of claim 23, wherein the donor plant is from the genus Zea and the acceptor plant is from the genus Triticum.
- 25. The method of claim 19, wherein the donor plant and the acceptor plant are from the same genus.
- 26. The method of claim 25, wherein the genus is selected from the group consisting of Zea, Triticum, Hordeum, Sorghum, Oryza, and Avena.
- 27. The method of claim 19, wherein the non-identical recombination sites are selected from the group consisting of FRT, mutant FRT, LOX, and mutant LOX sites.
- 28. The method of claim 27, wherein the recombinase is selected from the group consisting of FLP, Cre, and chimeric FLP/Cre.
- 29. The method of claim 27, wherein said sites are selected from the group consisting of FRT and mutant FRT sites.
- 30. The method of claim 29, wherein said mutant FRT sites are selected from the group consisting of FRT5, FRT6, and FRT7.
- 31. The method of claim 29, wherein said recombinase is FLP, or an active variant or fragment thereof.
- 32. The method of claim 19, wherein a nucleotide sequence encoding said recombinase is stably incorporated into the genome of the acceptor plant, and said nucleotide sequence encoding said recombinase is operably linked to a promoter.
- 33. The method of claim 19, wherein said target site further comprises a nucleotide sequence encoding said recombinase, said nucleotide sequence encoding said recombinase is operably linked to a promoter, and wherein the nucleotide sequence encoding the recombinase is located between the first and the second recombination sites.
- 34. The method of claim 19, wherein the target site further comprises a selectable marker located between the first and the second recombination sites.
- 35. The method of claim 19, wherein the DNA construct comprising the transfer cassette further comprises a selectable marker located between the first and the second recombination sites.
- 36. The method of claim 35, wherein the selectable marker is not operably linked to a promoter, and wherein the target site further comprises a promoter active in said plant, such that the promoter is operably linked to the first recombination site of the target site, whereby insertion of the selectable marker at the target site results in the selectable marker being operably linked to the promoter.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. application Ser. No. 09/850,492 filed May 7, 2001, and claims the benefit of U.S. application Ser. No. 60/203,056 filed May 8, 2000, now abandoned, which are all herein incorporated in entirety by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60203056 |
May 2000 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09850492 |
May 2001 |
US |
Child |
10895878 |
Jul 2004 |
US |