Stress tolerant cotton plants

• US 20090013431A1
• Filed: 10/27/2005
• Published: 01/08/2009
• Est. Priority Date: 10/29/2004
• Status: Active Grant

• Current Assignee: Bayer Cropscience NV (Bayer AG)
• Original Assignee: Bayer Bioscience NV (Bayer AG)
• Inventors: Van Thournout, Michel, Jacobs, John, Reynaerts, Arlette
• Granted Patent: US 8,053,636 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C12N 15/8271 for stress resistance, e.g. heavy metal resistance
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance

Abstract

The invention relates to the use of cotton parp2 gene or cDNA sequences to obtain stress tolerant cotton plants. Various cotton parp2 sequences are also provided.

Claims

1. A method of producing a stress tolerant cotton plant comprising the steps of

a) introducing a chimeric gene into a cotton cell, to generate a transgenic cotton cell, said chimeric gene comprising the following operably linked DNA fragments:

i) a plant expressible promoter;

ii) a transcribable DNA region comprising

(1) a first DNA region comprising a nucleotide sequence of at least 19 out of 20 consecutive nucleotides selected from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species;

(2) a second DNA region comprising a nucleotide sequence of at least 19 out of 20 consecutive nucleotides from said first DNA region;

whereby said first DNA region and said second DNA region are in an inverted repeat orientation with each other and wherein an RNA molecule transcribed from said transcribable region is capable of forming a double stranded RNA region between an RNA region transcribed from said first DNA region and an RNA region transcribed from said second DNA region; and

iii) a DNA region comprising a transcription termination and polyadenylation signal functional in plants;

b) regenerating said transgenic cotton cell to obtain a transgenic cotton plant; and

c) identifying a transgenic cotton plant which is more resistant to abiotic stress conditions than an untransformed cotton plant.

2. The method according to claim 1, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises the nucleotide sequence of any one of SEQ ID No.:

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

18, SEQ ID No.;

19. or SEQ ID No.;

20.

3. The method according to claim 1, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

13, of SEQ ID No.;

21, or SEQ ID No.;

22.

4. The method according to claim 1, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

15.

5. The method according to claim 1, wherein said first and said second DNA region comprise at least 50 consecutive nucleotides.

6. The method according to claim 1, wherein said first and said second DNA region comprise at least 200 consecutive nucleotides.

7. The method according to claim 1, wherein said transgenic cotton plant is identified using a fiber tissue culture assay.

8. The method according to claim 1, wherein said transgenic cotton plant is identified using a cold germination assay.

9. The method according to claim 1, wherein said transgenic cotton plant is identified by determination of the concentration of any one of reactive oxygen species, NAD or ATP.

20. The method according to claim 1 further comprising the step of crossing said stress resistant cotton plant with another cotton plant to obtain stress resistant cotton progeny plants.

10. A method of producing a stress tolerant cotton plant comprising the steps of:

a) providing one or more double stranded RNA molecules to cells of cotton plants, wherein said double stranded RNA molecules comprise two RNA strands, one RNA strand consisting essentially of an RNA nucleotide sequence of 19 out of 20 to 21 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species; and

b) identifying a cotton plant comprising said double stranded RNA molecule or molecules which is more resistant to abiotic stress conditions than a same cotton plant which does not comprise said double stranded RNA molecule or molecules.

11. The method according to claim 10, wherein said double stranded RNA is provided to said cells by integrating a chimeric gene into the genome of a said cell, said chimeric gene comprising the following operably linked DNA fragments:

a) a plant expressible promoter;

b) a DNA region comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in antisense orientation; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

12. The method according to claim 10, wherein said double stranded RNA is provided to said cells by integrating a chimeric gene into the genome of said cells, said chimeric gene comprising the following operably linked DNA fragments:

a) a plant expressible promoter;

b) a DNA region comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

13. The method according to claim 10, wherein said double stranded RNA is provided to said cells by integrating a chimeric gene into the genome of said cells, said chimeric gene comprising the following operably linked DNA fragments:

a) a plant expressible promoter;

b) a transcribable DNA region comprising

i) a first DNA region comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation;

ii) a second DNA region comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation, whereby an RNA molecule produced by transcription of said transcribed DNA region is capable of forming a double stranded RNA region by base-pairing between an RNA region corresponding to said first DNA region and an RNA region corresponding to said second RNA region; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

14. The method according to claim 10 wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises the nucleotide sequence of any one of SEQ ID No.:

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

18, SEQ ID No.;

19 or SEQ ID No.;

20.

15. The method according to claim 10, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

13, SEQ ID No.;

21, or SEQ ID No.;

22.

16. The method according to claim 10, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

15.

17. A method of producing to produce a stress tolerant cotton plant comprising the steps of

a) identifying a fragment of a PARP2 encoding DNA sequence from genomic DNA or cDNA obtainable from a cotton species or from a cotton species related to a cotton progenitor species using:

i) a DNA fragment comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13 as a probe;

ii) a DNA fragment comprising the nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

19, or SEQ ID No.;

20 as a probe;

iii) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 1382 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13 as a probe;

iv) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 2000 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID Nos;

21 or 22 as a probe

v) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 2000 consecutive nucleotides from a nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

19, or SEQ ID No.;

20 as a probe;

vi) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13 as a primer in a PCR reaction;

vii) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID Nos;

21 or 22 as a primer in a PCR reaction;

viii) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from the nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

19, or SEQ ID No.;

20 as a primer in a PCR reaction

ix) an oligonucleotide including the nucleotide sequence of any one of SEQ ID No.;

1, SEQ ID No.;

2, SEQ ID No.;

3, SEQ ID No.;

4, SEQ ID No.;

16, or SEQ ID No.;

17 as a primer in a PCR reaction;

or

x) a fragment which can be amplified from cotton genomic DNA or cDNA using as primers an oligonucleotide as described in vi, vii, viii or ix as a probe

b) providing one or more double stranded RNA molecules to cells of said cotton plants, wherein said double stranded RNA molecules comprise two RNA strands, one RNA strand consisting essentially of an RNA nucleotide sequence of 19 out of 20 to 21 consecutive nucleotides from the nucleotide sequence of said fragment of a PARP2 encoding DNA sequence; and

c) identifying a cotton plant comprising said double stranded RNA molecule or molecules which is more resistant to abiotic stress conditions than a same cotton plant which does not comprise said double stranded RNA molecule or molecules.

18. The method of claim 17, wherein the step of providing is carried out by providing to said plant cell a double stranded RNA molecule comprising a first nucleotide sequence of at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of said fragment of a PARP2 encoding DNA sequence, and a second nucleotide sequence which is the complement of said first nucleotide sequence.

19. The method of claim 17, wherein said double stranded RNA molecule or molecules is provided by integrating into the plant genome a chimeric DNA, said chimeric DNA comprising the following operably linked DNA fragments:

a) a plant expressible promoter;

b) a transcribable DNA region comprising

i) a first DNA region comprising a nucleotide sequence of at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of said fragment of a PARP2 encoding DNA sequence;

ii) a second DNA region comprising a nucleotide sequence of at least 19 consecutive nucleotides from said first DNA region;

whereby said first DNA region and said second DNA region are in an inverted repeat orientation with each other and wherein an RNA molecule transcribed from said transcribable region is capable of forming a double stranded RNA region between an RNA region transcribed from said first DNA region and an RNA region transcribed from said second DNA region; and

iii) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

54. The method of claim 17, wherein the cotton species is Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum, and the cotton species related to a cotton progenitor species is Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides.

55. The method of claim 17, wherein the fragment which can be amplified from cotton genomic DNA or cDNA is a fragment comprising the nucleotide sequence of SEQ ID No.:

18.

21. A method of identifying cotton parp2 DNA fragments comprising the steps of

a) providing genomic DNA or cDNA obtainable from a cotton species or from a cotton species related to a cotton progenitor species;

and

b) identifying said fragment by performing PCR using said genomic DNA or said cDNA and a primer, wherein said primer is (i) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13, (ii) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID Nos;

21 or 22, (iii) an oligonucleotide sequence including a nucleotide sequence comprising between 20 to 200 consecutive nucleotides from the nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

19, or SEQ ID No.;

20;

or (iv) an oligonucleotide including the nucleotide sequence of any one of SEQ ID No.;

1, SEQ ID No.;

2, SEQ ID No.;

3. SEQ ID No.;

4, SEQ ID No.;

16 or SEQ ID No.;

17;

or

identifying said fragment by performing hybridization using said genomic DNA or said cDNA and a probe, said probe is (i) a DNA fragment comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13;

(ii) a DNA fragment comprising the nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6. SEQ ID No.;

7. SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11. SEQ ID No.;

12, SEQ ID No.;

19 or SEQ ID No.;

20, (iii) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 1382 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.;

13;

(iv) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 2000 consecutive nucleotides from a nucleotide sequence encoding the amino acid sequence of SEQ ID Nos;

21 or 22 (v) a DNA fragment or oligonucleotide comprising a nucleotide sequence of between 20 to 2000 consecutive nucleotides from a nucleotide sequence of any one of SEQ ID No.;

5, SEQ ID No.;

6. SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

19 or SEQ ID No.;

20;

or (vi) a fragment comprising the nucleotide sequence of SEQ ID No.;

18

22. A method of isolating a cotton parp2 DNA fragment comprising the steps of

a) identifying said cotton parp2 fragment according to the method of claim 21;

and

b) isolating said cotton pa rp2 fragment.

23. A method of identifying cotton parp2 alleles correlated with increased stress tolerance comprising the steps of

a) providing a population of different cotton plant lines or plant lines related to cotton progenitor plants;

b) identifying in each plant line of said population a parp2 allele according to the method of claim 21;

c) analyzing the stress resistance of each plant line of said population and identifying those cotton plant lines;

d) correlating the increased stress resistance in a plant line to the presence of a specific parp2 allele.

24. A method of obtaining stress tolerant cotton plants, comprising the steps of

a) identifying a cotton parp2 allele correlated with increased stress tolerance according to the method of claim 23;

b) introducing said cotton parp2 allele into a cotton plant line of choice.

31. An isolated DNA fragment obtainable by the method of claim 21.

40. A cotton plant obtained by the method of claim 24.

56. The method of claim 21, wherein the cotton species is Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum, and the cotton species related to a cotton progenitor species is Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides.

57. The method of claim 23, wherein the population is a mutagenized population.

25. A method of identifying a stress resistant cotton plant comprising the following steps

a) initiate a fiber tissue culture from said cotton plant;

b) subject said fiber tissue culture to a stress condition; and

c) compare fiber initation or elongation in said culture to fiber initiation or elongation in a culture initated from a control plant and subject to said stresses conditions.

26. The method according to claim 25, wherein said stress condition comprises subjecting said fiber tissue culture to increased temperature for a period of time.

27. The method according to claim 26, wherein said increased temperature is selected from the range of 45 to 50°

C., and said period of time is in the range of 2 to 4 hours.

28. An isolated DNA fragment encoding a protein comprising the amino acid sequence of SEQ ID No.:

13, SEQ ID No.;

15, SEQ ID No.;

21, or SEQ ID No.;

22.

29. An isolated DNA fragment encoding a protein comprising the amino acid sequence of SEQ ID No.:

15.

30. An isolated DNA fragment comprising the nucleotide sequence of any one of the nucleotide sequences of SEQ ID No.:

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

18, SEQ ID No.;

19, or SEQ ID No.;

20.

32. A chimeric gene comprising the following operably linked DNA fragments:

a) a plant expressible promoter;

b) a transcribable DNA region comprising

i) a first DNA region comprising at least 20 consecutive nucleotides from the nucleotide sequence of a parp2 gene, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation;

ii) a second DNA region comprising at least 20 consecutive nucleotides from the nucleotide sequence of a parp2 genes, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation, whereby an RNA molecule produced by transcription of said transcribed DNA region is capable of forming a double stranded RNA region by base-pairing between an RNA region corresponding to said first DNA region and an RNA region corresponding to said second RNA region; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

35. The chimeric gene according to claim 32, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises the nucleotide sequence of any one of SEQ ID No.:

5, SEQ ID No.;

6, SEQ ID No.;

7, SEQ ID No.;

8, SEQ ID No.;

9, SEQ ID No.;

10, SEQ ID No.;

11, SEQ ID No.;

12, SEQ ID No.;

18, SEQ ID No. 19, or SEQ ID No. 20.

36. The chimeric gene according to claim 32, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

13, SEQ ID No.;

20, or SEQ ID No.;

21.

37. The chimeric gene according to claim 32, wherein said nucleotide sequence of said parp2 gene or parp2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.:

15.

38. A cotton plant cell comprising the chimeric DNA of claim 32.

39. A cotton plant consisting essentially of the cotton plant cells of claim 38.

41. A seed of a cotton plant according to claim 39.

33. A chimeric gene comprising the following operably linked DNA Fragments

a) a plant expressible promoter;

b) a DNA region comprising at least 20 consecutive nucleotides from the nucleotide sequence of a parp2 genes, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in sense orientation; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

34. A chimeric gene comprising the following operably linked DNA Fragments

a) a plant expressible promoter;

b) a DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide sequence of a parp2 genes, parp2 cDNA from a cotton species, or from a species related to a cotton progenitor species in antisense orientation; and

c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)

50. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)