METHODS FOR GENERATING TRANSGENIC PLANTS

• US 20140366223A1
• Filed: 06/06/2014
• Published: 12/11/2014
• Est. Priority Date: 06/11/2013
• Status: Active Grant

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Li, Xianggan, Elumalai, Sivamani
• Granted Patent: US 10,443,063 B2
• Time in Patent Office: Days
• US Class Current: 800/294
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers

Abstract

This invention provides a method for generating transgenic plants with a low copy number. Plant cells are transformed with polynucleotides containing transcriptional cassettes designed to trigger silencing of a gene which is essential for the plant cell to survive the transformation and regeneration process. The present invention enables the recovery of an increased number of transgenic plants which have only one copy of each desired transcriptional cassette.

Claims

1. A transformation method for making transgenic plant cells, comprising:

a. providing a first transcriptional cassette, wherein the first transcription cassette comprises a selectable marker;

b. providing a second transcriptional cassette comprising at least a DNA fragment of a gene which is essential for the plant cell to survive the transformation and regeneration process, wherein said fragment produces an RNA molecule;

c. providing at least one transcriptional cassette comprising a trait gene;

d. transforming said cassettes into a plurality of plant cells to produce a plurality of transgenic plant cells;

e. selecting for transgenic plant cells in the presence of a selection agent using the selectable marker of step (a); and

f. growing transgenic tissue from said transgenic plant cells of step (e),

wherein transgenic plant cells with two or more copies of the transcriptional cassette of step (b) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette is generated compared to methods without step (b).

2. The method of claim 1, wherein said DNA fragment of step (b) comprises at least 20, 30, 40, 50, 100, 200, 500, or 1000 consecutive polynucleotides of said gene which is essential for the plant cell to survive the transformation and regeneration process.

3. The method of claim 1, wherein said essential gene is ZmEPSPS, empty pericarp2, empty pericarp4, defective endosperm18, embryo defective 12, ameiotic1, ZmWHY1, or viviparous-5.

4. The method of claim 1, wherein said DNA fragment of step (b) has at least 60%, 70%, 80%, 85%, 90% or 95% sequence identity to a gene which is essential for the plant cell to survive the transformation and regeneration process, or a fragment thereof.

5. The method of claim 1, wherein said DNA fragment of step (b) is 19, 20, 21, 22, 23, or 24, or 25 consecutive polynucleotides of said gene.

6. The method of claim 1, wherein said DNA fragment of step (b) comprises SEQ ID NO:

12, SEQ ID NO;

14, or SEQ ID NO;

15.

7. The method of claim 6, wherein said DNA fragment has at least 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:

12, SEQ ID NO;

14, or SEQ ID NO;

15.

8. The method of claim 1, wherein the transcriptional cassette of step (a) and the transcriptional cassette of step (b) are the same.

9. The method of claim 1, wherein said DNA fragment of step (b) is a fragment of the selectable marker used in step (a).

10. The method of claim 9, wherein said selectable marker provides selection in the presence of a selection agent, wherein the selection agent is selected from the group comprising antibiotics, herbicides, drugs, metabolite analogs, metabolic intermediates, or phytohormone precursors.

11. The method of claim 10, wherein said selectable marker is a polynucleotide sequence encoding the protein PMI.

12. The method of claim 1, wherein said transgenic plant cells are generated by an Agrobacterium-mediated plant transformation process, wherein said transcriptional cassettes are incorporated in the T-DNA polynucleotide molecule.

13. The method of claim 1, wherein said first and second transcriptional cassettes are incorporated in a DNA plasmid and the DNA plasmid is transformed into a plurality of plant cells.

14. The method of claim 1, further comprising the step of determining the copy number of the transcriptional cassette encoding the selectable marker of step (a) and/or the copy number of the trait gene of step (c) comprised in said transgenic tissue.

15. The method of claim 14, further comprising the step of selecting a plant cell comprising a single copy of each transcriptional cassette and growing the plant cell into a plant.

16. A plant, plant tissue, or plant cell made by the method of claim 1.

17. A transformation method for making transgenic plant cells, comprising:

a. providing a first transcriptional cassette, wherein the first transcriptional cassette comprises a gene which is a mutated version or homologue of a native gene, wherein expression of the mutated transgene or homologue acts as a selectable marker;

b. providing at least one transcriptional cassette encoding a trait gene;

c. transforming said cassettes into a plurality of plant cells to produce a plurality of transgenic plant cells;

d. selecting for transgenic plant cells in the presence of a selection agent using the selectable marker of step (a); and

e. growing transgenic tissue from said transgenic plant cells of step (d);

wherein transgenic plant cells with two or more copies of the transcriptional cassette of step (a) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette is generated compared to methods without step (a).

18. The method of claim 17, further comprising the step of determining the copy number of each transcriptional cassette encoding a selectable marker of step (a) and/or a trait gene of step (b) comprised in said transgenic tissue.

19. The method of claim 17, wherein said mutated version or homologue of step (a) has at least 60%, 70%, 80%, 85%, 90% or 95% sequence identity to a native gene which is essential for the plant cell to survive the transformation and regeneration process.

20. The method of claim 17, wherein the transcriptional cassettes are incorporated in the same DNA plasmid and the DNA plasmid is transformed into a plurality of plant cells.

21. The method of claim 17, wherein the transgenic plant cells are generated by an Agrobacterium-mediated plant transformation process, wherein the T-DNA polynucleotide molecule comprises said transcriptional cassettes.

22. The method of claim 21, further comprising the step of selecting a plant cell comprising a single copy of the transcriptional cassette(s) and growing the plant cell into a plant.

23. The method of claim 17, wherein the first transcriptional cassette comprises a mutated version or homologue of a nucleotide sequence encoding EPSPS, wherein the polypeptide produced from said nucleotide sequence is not a target of glyphosate.

24. A plant, plant tissue, or plant cell made by the method of claim 17.

25. A transformation method for making transgenic plant cells, comprising:

a. providing a first transcriptional cassette, wherein a first transcriptional cassette comprises a modified heterologous EPSPS gene whose expression confers tolerance to the selection agent glyphosate;

b. providing at least one transcriptional cassette encoding a trait gene;

c. transforming said cassettes into a plurality of plant cells to produce a plurality of transgenic plant cells;

d. selecting for transgenic plant cells using glyphosate; and

e. growing transgenic tissues from said transgenic plant cells of step (d)

wherein said transgenic plant cells with two or more copies of the transcriptional cassette of step (a) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette is generated compared to methods without step (a).

26. The method of claim 25, wherein said modified heterologous EPSPS gene of step (a) has at least 60%, 70%, 80%, 85%, 90% or at least 95% sequence identity to the native EPSPS gene of the plant cells being transformed.

27. The method of claim 25, wherein said first transcriptional cassette comprises a fragment of said modified heterologous EPSPS gene, said fragment is 19, 20, 21, 22, 23, or 24, or 25 consecutive polynucleotides of said gene.

28. The method of claim 25, wherein the sequence of the first transcriptional cassette is selected from the group comprising SEQ ID NO:

1, SEQ ID NO;

4, or SEQ ID NO;

5.

29. The method of claim 25, wherein the heterologous EPSPS gene of step (a) comprises SEQ ID NO:

12 or SEQ ID NO;

16.

30. The method of claim 25, wherein the first and second transcriptional cassettes are incorporated in the same DNA plasmid and the DNA plasmid is transformed into a plurality of plant cells.

31. The method of claim 25, wherein the transgenic plant cells are generated by an Agrobacterium-mediated plant transformation process, wherein the T-DNA polynucleotide molecule comprises said transcriptional cassettes.

32. The method of claim 25, further comprising the step of determining the copy number of each transcriptional cassette comprised in said transgenic tissues.

33. The method of claim 32, further comprising the step of selecting a transgenic tissue comprising a single copy of each transcriptional cassette and growing the plant tissue into a plant.

34. A plant, plant tissue, or plant cell made by the method of claim 25.

35. A transformation method for making transgenic plant, comprising:

a. providing a first transcriptional cassette, wherein the first transcriptional cassette comprises a 5′

regulatory sequence comprising a promoter operably linked at the 3′

end to at least 20 polynucleotides of an exon of a gene which is essential for the plant cell to survive the transformation and regeneration process, wherein said 5′

-regulatory sequence has been engineered to include a translation initiation sequence at the 3′

end of said 5′

-regulatory sequence;

b. providing at least one transcriptional cassette encoding a selectable marker;

c. providing at least one transcriptional cassette encoding a trait gene;

d. transforming said cassettes into a plurality of plant cells to produce a plurality of transgenic plant cells;

e. selecting for transgenic plant cells in the presence of a selection agent using the selectable marker of step (b); and

f. growing transgenic tissues from said transgenic plant cells of step (e),

wherein transgenic plant cells with two or more copies of the transcriptional cassette of step (a) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette is generated compared to methods without step (a).

36. The method of claim 35, wherein said 5′

regulatory sequence of the transcriptional cassette of step (a) further comprises an intron or linker operably linked at the 3′

end of said first exon, and at least 20 polynucleotides of the second exon of said essential gene operably linked at the 3′

end of said intron or linker.

37. The method of claim 35, wherein said 5′

regulatory sequence of the transcriptional cassette of step (a) has at least 60%, 70%, 80%, 85%, 90% or at least 95% sequence identity to a native gene which is essential for the plant cell to survive the transformation and regeneration process, or a fragment thereof.

38. The method of claim 35, wherein said 5′

regulatory sequence of the transcriptional cassette is SEQ ID NO;

6.

39. The method of claim 35, wherein a polynucleotide encoding a trait gene or a selectable marker is operably linked to said 5′

-regulatory sequence of step (a).

40. The method of claim 35, wherein the transcriptional cassette of step (a) and of step (b) is the same.

41. The method of claim 35, wherein the transcriptional cassettes are incorporated in the same DNA plasmid and said DNA plasmid is transformed into a plurality of plant cells.

42. The method of claim 35, wherein the transgenic plant cells are generated by an Agrobacterium-mediated plant transformation process, wherein the T-DNA polynucleotide molecule comprises said transcriptional cassettes.

43. The method of claim 35, further comprising the step of determining the copy number of each transcriptional cassette comprised in said transgenic tissues.

44. The method of claim 43, further comprising the step of selecting a transgenic tissue comprising a single copy of each transcriptional cassette and growing the plant tissue into a plant.

45. A plant, plant tissue, or plant cell made by the method of claim 35.

46. An Agrobacterium-mediated transformation method for making transgenic plant cells, comprising:

a. providing a first T-DNA polynucleotide comprising all transcriptional cassettes of interest, wherein the first transcriptional cassette comprises a selectable marker and at least one transcriptional cassette comprises a trait gene;

b. providing a second T-DNA polynucleotide comprising a transcriptional cassette, wherein said transcriptional cassette is transcribed into RNA and whose expression induces reduced expression or silencing of said selectable marker;

c. providing a DNA plasmid for Agrobacterium-mediated transformation, comprising said first T-DNA polynucleotide and said second T-DNA polynucleotide;

d. performing Agrobacterium-mediated transformation on a plurality of cells to produce a plurality of transgenic plant cells;

e. selecting for transgenic plant cells which only contain said first T-DNA polynucleotide using the selection agent for said selectable marker; and

f. growing transgenic tissues from said transgenic plant cells of step (e),

wherein transgenic plant cells with the second T-DNA polynucleotide of step (b) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette within the first T-DNA polynucleotide of step (a) is generated compared to methods without step (b).

47. The method of claim 46, wherein the transcriptional cassette of step (b) has at least 60%, 70%, 80%, 85%, 90% or at least 95% sequence identity to said selectable marker, or a fragment thereof.

48. The method of claim 46, wherein the second T-DNA polynucleotide is further comprised of a first polynucleotide fragment of the selectable marker, wherein said first polynucleotide fragment is at least 20 nucleotides in length, operably linked to a polynucleotide linker, operably linked to a second polynucleotide fragment at least 20 polynucleotides in length which is complementary to the first polynucleotide fragment.

49. The method of claim 46, wherein said selectable marker provides selection in the presence of a selection agent, wherein the selection agent is selected from the group comprising antibiotics, herbicides, drugs, metabolite analogs, metabolic intermediates, or phytohormone precursors.

50. The method of claim 46, wherein the selectable marker is PMI.

51. The method of claim 46, wherein said transcriptional cassette of step (b) comprises SEQ ID NO:

15.

52. A DNA plasmid comprising the first and second T-DNA polynucleotides of claim 46.

53. The method of claim 46, further comprising the step of determining the copy number of each transcriptional cassette of interest comprised in said transgenic tissues.

54. The method of claim 53, further comprising the step of selecting a transgenic tissue comprising a single copy of the transcriptional cassettes and growing the plant tissue into a plant.

55. A plant, plant tissue, or plant cell made by the method of claim 46.