Genetically engineered duckweed

• US 6,040,498 A
• Filed: 08/11/1998
• Issued: 03/21/2000
• Est. Priority Date: 08/11/1998
• Status: Expired due to Term

• Current Assignee: North Carolina State University (University of North Carolina System)
• Original Assignee: North Carolina State University (University of North Carolina System)
• Inventors: Stomp, Anne-Marie, Rajbhandari, Nirmala
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Mehta, Ashwin D.
• Application Number:
  US09/132,536
• Time in Patent Office: 588 Days
• Field of Search: 435/69.1, 435/320.1, 435/410, 435/413, 435/418, 435/419, 435/430, 435/430.1, 435/469, 435/69.4, 435/69.51, 435/69.6, 435/70.21, 536/23.6, 536/23.51, 536/23.52, 800/278, 800/288, 800/294, 800/295, 800/298, 800/300
• US Class Current: 800/294
• CPC Class Codes: A01H 13/00 Algae unicellular algae C12N1/12
• CPC Class Codes: A01H 4/005 Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon

Abstract

Methods and compositions for the efficient transformation of duckweed are provided. Preferably, the methods involve transformation by either ballistic bombardment or Agrobacterium. In this manner, any gene or nucleic acid of interest can be introduced and expressed in duckweed plants. Transformed duckweed plants, cells, tissues are also provided. Transformed duckweed plant tissue culture and methods of producing recombinant proteins and peptides from transformed duckweed plants are also disclosed.

Claims

1. A method for stably transforming duckweed tissue with a nucleotide sequence of interest, the method comprising the steps of:

(a) inoculating a duckweed plant tissue with Agrobacterium comprising a vector which comprises a nucleotide sequence of interest; and

(b) co-cultivating the tissue with the Agrobacterium to produce stably transformed tissue.

2. The method according to claim 1, further comprising the step of culturing the transformed tissue in a medium comprising an antibiotic sufficient to inhibit the growth of Agrobacterium.

3. The method according to claim 1, further comprising the step of culturing the transformed tissue in a medium comprising the selection agent.

4. The method according to claim 3, further comprising the step of regenerating stably transformed duckweed plants from the stably transformed tissue.

5. The method according to claim 1, wherein the tissue is callus tissue.

6. The method according to claim 1, wherein the tissue is meristematic tissue.

7. The method according to claim 1, wherein the tissue is frond tissue.

8. The method according to claim 1, wherein the duckweed tissue is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna.

9. The method according to claim 1, wherein the duckweed tissue is selected from the group consisting of Lemna minor, Lemna miniscula, and Lemna gibba.

10. The method according to claim 1, wherein the Agrobacterium is Agrobacterium tumefaciens.

11. The method according to claim 1, wherein the Agrobacterium is Agrobacterium rhizogenes.

12. The method according to claim 1, wherein the vector is a super-binary vector.

13. The method according to claim 1, wherein the vector is a C58-derived vector.

14. The method according to claim 1, wherein the gene which confers resistance to a selection agent is selected from the group consisting of neo, bar, pat, ALS, HPH, HYG, EPSP and Hm1.

15. The method according to claim 1, wherein the nucleotide sequence comprises two genes of interest.

16. The method according to claim 1, wherein the nucleotide sequence encodes a protein or peptide selected from the group consisting of insulin, growth hormone, α

-interferon, β

-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin, leptin, and serum albumin.

17. The method according to claim 1, wherein the nucleotide sequence encodes at least one protein or peptide subunit of a multimeric protein selected from the group consisting of hemoglobin, collagen, P450 oxidase, and a monoclonal antibody.

18. A stably transformed duckweed tissue culture produced according to the method of claim 3.

19. A stably transformed duckweed plant produced according to the method of claim 4.

42. The method according to claim 1, wherein the vector is not a super-binary vector.

43. The method according to claim 1, wherein the Agrobacterium comprises an inducible vir gene.

44. The method according to claim 1, wherein the vector comprises a Cauliflower Mosaic virus 35S promoter.

45. The method according to claim 1, wherein the vector is a pBIN19 derivative.

46. The method according to claim 5, wherein said co-cultivating step is carried out for a time of about 1 to about 3 days.

47. The method according to claim 1, wherein the vector comprises a mas promoter.

64. The method of claim 1, wherein said nucleotide sequence of interest is a gene which confers resistance to a selection agent.

65. The method of claim 1, wherein said nucleotide sequence of interest is a gene which confers resistance to a selection agent.

20. A stably transformed duckweed plant comprising a heterologous nucleic acid of interest incorporated in its genome wherein said plant is produced via an Agrobacterium-mediated method.

21. The stably transformed duckweed plant according to claim 20, wherein said heterologous nucleic acid of interest is flanked by T-DNA border sequences incorporated in its genome.

22. The stably transformed duckweed plant according to claim 21, wherein said duckweed plant comprises fewer than 5 copies of said heterologous nucleic acid of interest.

23. The stably transformed duckweed plant according to claim 20 or claim 21, wherein said duckweed plant is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna.

24. The stably transformed duckweed plant according to claim 21, wherein said duckweed plant is selected from the group consisting of Lemna minor, Lemna miniscula, and Lemna gibba.

25. The stably transformed duckweed plant according to claim 21, wherein said heterologous nucleic acid comprises at least one expression cassette comprising a gene which confers resistance to a selection agent.

26. The stably transformed duckweed plant according to claim 25, wherein said gene which confers resistance to a selection agent is of neo, bar, pat, ALS, HPH, HYG, EPSP and Hml.

27. The stably transformed duckweed plant according to claim 21, wherein said heterologous nucleic acid comprises two genes of interest.

28. The stably transformed duckweed plant according to claim 21, wherein said heterologous nucleic acid encodes a protein or peptide selected from the group consisting of insulin, growth hormone, α

-interferon, β

-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin, leptin, and serum albumin.

29. The stably transformed duckweed plant according to claim 21, wherein said heterologous nucleic acid encodes at least one protein or peptide subunit of a multimeric protein selected from the group consisting of hemoglobin, collagen, P450 oxidase, and a monoclonal antibody.

30. A method of producing recombinant proteins or peptides, comprising the steps of

(a) culturing a stably transformed duckweed plant that expresses at least one heterologous protein or peptide;

and

(b) collecting the at least one heterologous protein or peptide from the duckweed cultures.

31. The method according to claim 30, wherein the stably transformed duckweed plant is grown on wastewater.

32. The method according to claim 30, wherein the stably transformed duckweed plant expresses and assembles all of the subunits of a multimeric protein.

33. The method according to claim 32, wherein the multimeric protein is selected from the group consisting of collagen, hemoglobin, P450 oxidase, and a monoclonal antibody.

34. The method according to claim 30, wherein the stably transformed duckweed plant is grown in a bioreactor vessel.

35. The method according to claim 30, wherein one recombinant protein or peptide is produced.

36. The method according to claim 30, wherein the at least one heterologous protein or peptide is a therapeutic protein or peptide.

37. The method according to claim 30, wherein the at least one protein or peptide is selected from the group consisting of insulin, growth hormone, α

-interferon, β

-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin, leptin, and serum albumin.

38. The method according to claim 30, wherein the at least one heterologous protein or peptide is an enzyme.

39. The method according to claim 30, wherein the at least one heterologous protein or peptide is secreted from the stably transformed duckweed plant.

40. The stably transformed duckweed plant according to claim 30, wherein said duckweed plant is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna.

41. The stably transformed duckweed plant according to claim 30, wherein said duckweed plant is selected from the group consisting of Lemna minor, Lemna miniscula, and Lemna gibba.

48. A method for stably transforming duckweed tissue from the genus Lemna with a nucleotide sequence of interest, the method comprising the steps of:

(a) inoculating a duckweed plant tissue with Agrobacterium comprising a vector which comprises a nucleotide sequence of interest, wherein the duckweed plant tissue is from the genus Lemna; and

(b) co-cultivating the tissue with the Agrobacterium to produce stably transformed tissue.

49. The method according to claim 48, further comprising the step of regenerating stably transformed duckweed plants from the transformed tissue.

50. The method according to claim 48, wherein the duckweed plant tissue is from a species of Lemna minor.

51. The method according to claim 48, wherein the tissue is a callus tissue.

52. The method according to claim 51, wherein said co-cultivating step is carried out for a time of about 1 to about 3 days.

53. The method according to claim 48, wherein the vector is not a super-binary vector.

54. The method according to claim 48, wherein the Agrobacterium comprises an inducible vir gene.

55. The method according to claim 48, wherein the vector comprises a Cauliflower Mosaic virus 35S promoter.

56. The method according to claim 48, wherein the vector is a pBIN19 derivative.

57. The method according to claim 48, wherein the vector comprises a mas promoter.

58. A stably transformed duckweed plant produced according to the method of claim 48.

61. The stably transformed duckweed plant of claim 58, wherein said stably transformed duckweed plant is from a species of Lemna minor.

59. A stably transformed duckweed plant from the genus Lemna comprising a heterologous nucleic acid sequence incorporated in its genome wherein said plant is produced via an Agrobacterium-mediated method.

60. The stably transformed duckweed plant according to claim 59, wherein said heterologous nucleic acid sequence is flanked by T-DNA border sequences incorporated in its genome.

62. A method of producing recombinant proteins or peptides, comprising:

(a) culturing a stably transformed duckweed plant from the genus Lemna that expresses at least one heterologous protein or peptide; and

(b) collecting the at least one protein or peptide from the duckweed cultures.

63. The method of claim 62, wherein said stably transformed duckweed plant is a species of Lemna minor.