Claims
- 1. A method for producing a transgenic plant, comprising (a) agitating a solution comprising a germinating plant seedling, or explant thereof, and at least one Agrobacterium strain that harbors a plasmid vector carrying a desired polynucleotide; (b) cultivating said seedling to produce a plant; and (c) screening said plant to determine if said desired polynucleotide is integrated into the genome of at least one cell of said plant, wherein said plant is stably transformed, and wherein the step of agitating the solution does not comprise sonication.
- 2. The method of claim 1, wherein said germinating plant seedling is from a monocotyledenous plant.
- 3 The method of claim 2, wherein said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, rice, oat, barley, orchid, iris, lily, onion, sugarcane, and sorghum.
- 4. The method of claim 3, wherein said turfgrass is selected from the group consisting of Agrostis spp., Poa pratensis, Lolium spp., Festuca arundinacea, Festuca rubra commutata, Cynodon dactylon, Pennisetum clandestinum, Stenotaphrum secundatum, Zoysia japonica, and Dichondra micrantha.
- 5. The method of claim 1, wherein said germinating plant seedling is from a dicotyledenous plant.
- 6. The method of claim 5, wherein said dicotyledenous plant is selected from the group consisting of cotton, tobacco, Arabidopsis, tomato, potato, sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, and cactus.
- 7. The method of claim 1, wherein expression of said desired polynucleotide in said stably transformed plant confers a trait to said plant selected from the group consisting of increased drought tolerance, reduced height, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.
- 8. The method of claim 7, wherein said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5′-untranslated region of said gene, the 3′-untranslated region of said gene, the leader sequence associated with said gene, the trailer sequence associated with said gene, or combinations of such sequences, wherein the orientation of any of said sequences is either in the sense or antisense orientation.
- 9. The method of claim 8 wherein said desired polynucleotide expresses a peptide or protein that is an antifungal, a nutritional peptide or protein, a transcription factor, a receptor that binds to pathogen-derived ligands, a hemoglobin, an oxidase, an enzyme of the lignin biosynthesis pathway, an enzyme of industrial value, or an antigen.
- 10. The method of claim 1, wherein said desired polynucleotide comprises a gene operably linked to a promoter and a terminator.
- 11. The method of claim 10, wherein the sequences of said promoter and said terminator naturally occur in the genome of edible foods.
- 12. The method of claim 1, wherein said vector comprises (a) a T-DNA or a P-DNA that comprises (i) said desired polynucleotide, and (ii) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants; and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.
- 13. The method of claim 12, wherein desired polynucleotide comprises a gene operably linked to a promoter and a terminator.
- 14. The method of claim 12, wherein said backbone integration marker gene is operably linked to a promoter and a terminator.
- 15. The method of claim 14, wherein said backbone integration marker is a cytokinin gene.
- 16. The method of claim 12, wherein said cytokinin gene is IPT, and said plant is a dicotyledon plant.
- 17. The method of claim 12, wherein said backbone integration marker is PGA22, TZS, HOC1, CKI1, and ESR1.
- 18. The method of claim 12, wherein said border-like sequences of said P-DNA range in size from 20 to 100 bp and share between 52% and 96% sequence identity with a T-DNA border sequence from Agrobacterium tumafaciens.
- 19. The method of claim 12, wherein expression of said selectable marker gene confers fertilizer tolerance to said transgenic plant and progeny thereof.
- 20. The method of claim 19, wherein said selectable marker gene confers resistance to cyanamide.
- 21. The method of claim 20, wherein said selectable marker gene is selected from the group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H1, and SEQ ID NO. 1.
- 22. The method of claim 12, wherein said selectable marker gene is operably linked to a yeast ADH terminator.
- 23. The method of claim 12, wherein said selectable marker gene is an antibiotic resistance gene.
- 24. The method of claim 23, wherein said antibiotic resistance gene is selected from the group consisting of hygromycin phosphotransferase, neomycin phosphotransferase, streptomycin phosphotransferase, and bleomycin-binding protein.
- 25. The method of claim 12, wherein said selectable marker gene is a herbicide resistance gene.
- 26. The method of claim 25, wherein said herbicide resistance gene is selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, and phosphinothricin acetyl transferase.
- 27. The method of claim 1, wherein the step of agitating the solution is accomplished by vortexing.
- 28. The method of claim 27, wherein said solution is vortexed from about 60 seconds to several hours.
- 29. The method of claim 28, wherein said solution is vortexed for about 5 minutes to about 30 minutes.
- 30. The method of claim 1, wherein the step of cultivating said seedling to produce a transgenic plant comprises transferring said Agrobacterium-transformed seedling to soil, and exposing said transformed seedling to conditions that promote growth.
- 31. The method of claim 1, wherein the step of cultivating said seedling to produce transgenic plants comprises cultivating said Agrobacterium-transformed seedling in or on tissue culture medium prior to transferring said transformed seedling to soil, and exposing said transformed seedling to conditions that promote growth.
- 32. The method according to claim 31, further comprising (i) producing a callus from said transformed seedling cultivated on tissue culture medium; and (ii) inducing shoot and root formation from said callus, prior to transferring to soil.
- 33. The method of claim 32, wherein said vector comprises (a) a T-DNA or a P-DNA that comprises (i) said desired polynucleotide, and (ii) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants; and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA, or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.
- 34. The method of claim 32, wherein the step of producing a callus from said transformed seedling comprises (i) transferring said transformed seedling to tissue culture media that contains auxin and cyanamide; (ii) selecting fertilizer-tolerant calli; (iii) inducing shoot and root formation from said calli; and (iv) transferring calli with shoots and roots to soil and exposing said calli to conditions that promote growth of said transgenic plants from said calli.
- 35. The method of claim 1, wherein said transformed plant seedling is grown to maturity, crossed to a non-transformed plant and said desired polynucleotide transmitted to at least one progeny plant.
- 36. The method of claim 1, wherein said transformed plant seedling is grown to maturity, selfed, and said desired polynucleotide transmitted to progeny.
- 37. A vector, which can be maintained in Agrobacterium, comprising: (a) a T-DNA or a P-DNA that comprises (i) a desired polynucleotide, and (ii) a selectable marker gene that is operably linked to a terminator not naturally expressed in plants, and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.
- 38. The vector of claim 37, wherein desired polynucleotide is operably linked to a promoter and a terminator.
- 39. The vector of claim 37, wherein said backbone integration marker gene is operably linked to a promoter and a terminator.
- 40. The vector of claim 39, wherein said backbone integration marker is a cytokinin gene.
- 41. The vector of claim 40, wherein said cytokinin gene is IPT.
- 42. The vector of claim 37, wherein said backbone integration marker is selected from the group consisting of PGA22, TZS, HOC1, CKI1, and ESR1.
- 43. The vector of claim 37, wherein expression of said selectable marker gene confers fertilizer tolerance to said transgenic plants and progeny thereof.
- 44. The vector of claim 43, wherein said selectable marker gene that confers fertilizer tolerance is a selectable marker gene that confers resistance to cyanamide.
- 45. The vector of claim 44, wherein said selectable marker gene that confers resistance to cyanamide is selected from the group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H1, and SEQ ID NO. 1.
- 46. The vector of claim 37, wherein said selectable marker gene is an antibiotic resistance gene.
- 47. The vector of claim 46, wherein said antibiotic resistance gene is selected from the group consisting of hygromycin phosphotransferase, neomycin phosphotransferase, streptomycin phosphotransferase, and bleomycin-binding protein.
- 48. The vector of claim 37, wherein said selectable marker gene is a herbicide resistance gene.
- 49. The vector of claim 48, wherein said herbicide resistance gene is selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, and phosphinothricin acetyl transferase.
- 50. The vector of claim 44, wherein said selectable marker gene encodes a protein comprising the sequence of SEQ ID NO. 1.
- 51. The vector of claim 38, wherein all of the genetic elements in said vector that are intended for transfer to plant cells are isolated from edible foods.
- 52. The vector of claim 38, wherein said promoter and said terminator naturally occur in the genome of edible food sources.
- 53. The vector of claim 37, wherein said desired polynucleotide comprises a plant gene derived from the genome of an edible food source.
- 54. The vector of claim 37, wherein expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genomes, wherein said trait is selected from the group consisting of increased drought tolerance, reduced height, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.
- 55. The vector of claim 54, wherein said desired polynucleotide comprises a gene that encodes a peptide or protein that is an antifungal, a nutritional peptide or protein, a transcription factor, a receptor that binds to pathogen-derived ligands, a hemoglobin, an oxidase, an enzyme of the lignin biosynthesis pathway, an enzyme of industrial value, or an antigen.
- 56. A method for producing a transgenic plant, comprising: (A) infecting plant tissue with an Agrobacterium transformation vector that comprises (i) a T-DNA or a P-DNA that comprises (a) said desired polynucleotide, and (b) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants; and (ii) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA; (B) cultivating said seedling to produce plants; and (C) screening said plants for stable integration of said desired polynucleotide.
- 57. The method of claim 56, wherein said plant tissue is a germinating plant seedling.
- 58. The method of claim 56, wherein desired polynucleotide is operably linked to a promoter and a terminator.
- 59. The method of claim 56, wherein said backbone integration marker gene is operably linked to a promoter and a terminator.
- 60. The method of claim 59, wherein said backbone integration marker is a cytokinin gene.
- 61. The method of claim 60, wherein said cytokinin gene is IPT.
- 62. The method of claim 56, wherein said backbone integration marker is PGA22, TZS, HOC1, CKI1, and ESR1.
- 63. The method of claim 56, wherein expression of said selectable marker gene confers fertilizer tolerance to said transgenic plants and progeny thereof.
- 64. The method of claim 63, wherein said selectable marker gene that confers fertilizer tolerance is a selectable marker gene that confers resistance to cyanamide.
- 65. The method of claim 64, wherein said selectable marker gene that confers resistance to cyanamide is selected from said group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H1, and SEQ ID NO. 1.
- 66. The method of claim 56, wherein the step of cultivating said seedling comprises (i) transferring said Agrobacterium-transformed seedling to soil and exposing said transformed seedling to conditions that promote growth.
- 67. The method of claim 56, wherein the step of screening said plants for stable integration of said desired polynucleotide comprises (i) exposing said plants to a screening solution containing a substance that only plants that express said selectable marker gene are tolerant to; (ii) growing said plants to maturity and allowing said plants to produce T1 seedling; (iii) transferring said T1 seedling to soil; and (iv) exposing said seedling to said screening solution.
- 68. The method of claim 56, wherein the step of infecting said germinating plant seedling comprises submerging said seedling into a solution comprising an Agrobacterium strain that contains said Agrobacterium transformation vector; and (b) vortexing said solution.
- 69. The method of claim 56, wherein said selectable marker gene is an antibiotic resistance gene.
- 70. The method of claim 69, wherein said antibiotic resistance gene is selected from the group consisting of hygromycin phosphotransferase, neomycin phosphotransferase, streptomycin phosphotransferase, and bleomycin-binding protein.
- 71. The method of claim 56, wherein said selectable marker gene is a herbicide resistance gene.
- 72. The method of claim 71, wherein said herbicide resistance gene is selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, and phosphinothricin acetyl transferase.
- 73. The method of claim 63, wherein said selectable marker gene is operably linked to a yeast ADH terminator.
- 74. The method of claim 58, wherein said promoter and said terminator naturally occur in the genome of a food source.
- 75. The method of claim 56, wherein said desired polynucleotide is derived from the genome of a food source.
- 76. The method of claim 56, wherein expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genomes, wherein said trait is selected from the group consisting of increased drought tolerance, reduced height, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.
- 77. The method of claim 76, wherein said desired polynucleotide encodes a peptide or protein that is an antifungal, a nutritional peptide or protein, a transcription factor, a receptor that binds to pathogen-derived ligands, a hemoglobin, an oxidase, an enzyme of the lignin biosynthesis pathway, an enzyme of industrial value, or an antigen.
- 78. The method of claim 67, wherein said substance contained in said screening solution is hydrogen cyanamide.
- 79. A method of modifying the expression of a functional gene in a plant cell comprising:
(a) constructing a first T-DNA or P-DNA that comprises a desired polynucleotide that is capable of modifying the expression of a functional gene in a plant cell; (b) constructing a second T-DNA or P-DNA that comprises a selectable marker gene operably linked to a promoter and terminator, wherein said terminator does not naturally occur in plants; (c) exposing germinating plant seedlings to one or more Agrobacterium strains that contain said first T-DNA or P-DNA and said second T-DNA or P-DNA; (d) selecting only those transformed seedlings that transiently express said selectable marker gene; and (e) selecting from the seedlings of (d), a seedling that comprises in its genome said desired polynucleotide but not said selectable marker; wherein expression of said desired polynucleotide in the seedling of (e) modifies the expression of a functional gene in a plant cell in said seedling.
- 80. The method of claim 79, wherein said seedlings are from a monocotyledenous plant.
- 81. The method of claim 80, wherein said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, rice, oat, barley, orchid, iris, lily, onion, sugarcane, and sorghum.
- 82. The method of claim 79, wherein said plant seedling is from a dicotyledenous plant.
- 83. The method of claim 82, wherein said dicotyledenous plant is selected from the group consisting of potato, tobacco, tomato, sugar beet, broccoli, cassava, sweet potato, pepper, cotton, poinsettia, legumes, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, and cactus.
- 84. The method of claim 79, wherein a first vector carries said first T-DNA or P-DNA and a second vector carries said second T-DNA or P-DNA.
- 85. The method of claim 79, wherein a first vector carries said first P-DNA and a second vector carries said second P-DNA.
- 86. The method of claim 84, wherein said second polynucleotide comprises at least one of an omega-mutated virD2 polynucleotide, a codA polynucleotide, and a codA::upp fusion polynucleotide.
- 87. A plant made by the method of claim 79.
- 88. A method for producing a transgenic plant, comprising: (A) infecting a germinating plant seedling with an Agrobacterium transformation vector that comprises (i) a T-DNA or a P-DNA that comprises (a) said desired polynucleotide, and (b) a gene operably linked to a terminator that is not naturally expressed in plants, wherein said gene confers fertilizer tolerance to plants in which it is expressed; and (ii) a cytokinin gene, wherein said desired polynucleotide and said selectable marker gene are flanked by said border sequences of said T-DNA or by said border-like sequences of said P-DNA; (B) transferring said transformed seedling to soil and allowing them to grow into plants; (C) exposing said plants to 0.05% to 20% hydrogen cyanamide.
- 89. The method of claim 88, wherein said fertilizer tolerance gene confers resistance to cyanamide.
- 90. The method of claim 89, wherein said selectable marker gene that confers resistance to cyanamide is selected from the group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H7, and SEQ ID NO. 1.
- 91. A method for producing a transgenic plant, comprising (a) vortexing a solution comprising a germinating plant seedling and at least one Agrobacterium strain that harbors a vector carrying a desired polynucleotide; (b) transferring said Agrobacterium-transformed seedling to soil, and exposing said transformed seedling to conditions that promote growth; and (d) screening said plants to determine if said desired polynucleotide is integrated into the genome of at least one cell of said plant, wherein a plant comprising said desired polynucleotide in said genome is a transgenic plant.
- 92. The method of claim 91, wherein said germinating plant seedling are from a monocotyledenous plant.
- 93. The method of claim 92, wherein said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, rice, oat, barley, orchid, iris, lily, onion, sugarcane, and sorghum.
- 94. The method of claim 93, wherein said turfgrass is selected from the group consisting of Agrostis spp., Poa pratensis, Lolium spp., Kentucky Bluegrass And Perennial Ryegrass Mix; Festuca arundinacea, Festuca rubra commutata, Cynodon dactylon, Pennisetum clandestinum, Stenotaphrum secundatum, Zoysia japonica, and Dichondra micrantha.
- 95. The method of claim 91, wherein said germinating plant seedling are from a dicotyledenous plant.
- 96. The method of claim 95, wherein said dicotyledenous plant is selected from the group consisting of cotton, tobacco, Arabidopsis, tomato, potato sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium and cactus.
- 97. The method of claim 91, wherein expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genome, wherein said trait is selected from the group consisting of increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.
- 98. The method of claim 91, wherein said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5′-untranslated region of said gene, the 3′-untranslated region of said gene, the leader sequence associated with said gene, or the trailer sequence associated with said gene, or combinations of such sequences, wherein the orientation of any of said sequences is either in the sense or antisense orientation.
- 99. The method of claim 8 wherein said desired polynucleotide expresses a peptide or protein that is an antifungal, a nutritional peptide or protein, a transcription factor, a receptor that binds to pathogen-derived ligands, a hemoglobin, an oxidase, an enzyme of the lignin biosynthesis pathway, an enzyme of industrial value, or an antigen.
- 100. The method of claim 98, wherein said desired polynucleotide is operably linked to a promoter and a terminator.
- 101. The method of claim 100, wherein the sequences of said promoter and said terminator naturally occur in the genome of a food source.
- 102. The method of claim 100, wherein said vector comprises (a) a T-DNA or a P-DNA that comprises (i) said desired polynucleotide, and (ii) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants; and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.
- 103. The method of claim 102, wherein said backbone integration marker gene is operably linked to a promoter and a terminator.
- 104. The method of claim 103, wherein said backbone integration marker is a cytokinin gene.
- 105. The method of claim 104, wherein said cytokinin gene is IPT, and said plant is a dicotyledon plant.
- 106. The method of claim 102, wherein said backbone integration marker is PGA22, TZS, HOC1, CKI1, and ESR1.
- 107. The method of claim 91, wherein the step of screening comprises detecting the presence of said desired polynucleotide in cells of said transgenic plant.
- 108. The method of claim 91, further comprising producing progeny from said transgenic plant and detecting the presence of said desired polynucleotide in cells of said progeny.
- 109. The method of claim 102, wherein said border-like sequences of said P-DNA range in size from 20 to 100 bp and share between 52% and 96% sequence identity with a T-DNA border sequence from Agrobacterium tumafaciens.
- 110. The method of claim 102, wherein expression of said selectable marker gene confers fertilizer tolerance to said transgenic plants and progeny thereof.
- 111. The method of claim 102, wherein said selectable marker gene that confers fertilizer tolerance is a selectable marker gene that confers resistance to cyanamide.
- 112. The method of claim 111, wherein said selectable marker gene that confers resistance to cyanamide is selected from the group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H1, and SEQ ID NO. 1.
- 113. The method of claim 102, wherein said selectable marker gene is operably linked to a yeast ADH terminator.
- 114. The method of claim 102, wherein said selectable marker gene is an antibiotic resistance gene.
- 115. The method of claim 114, wherein said antibiotic resistance gene is selected from the group consisting of hygromycin phosphotransferase, neomycin phosphotransferase, streptomycin phosphotransferase, and bleomycin-binding protein.
- 116. The method of claim 102, wherein said selectable marker gene is a herbicide resistance gene.
- 117. The method of claim 116, wherein said herbicide resistance gene is selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, and phosphinothricin acetyl transferase.
- 118. The method of claim 91, wherein said solution is vortexed from about 60 seconds to several hours.
- 119. The method of claim 91, wherein said solution is vortexed for about 5 minutes to about 30 minutes.
- 120. A method for producing a transgenic plant, comprising (a) vortexing a solution comprising a germinating plant seedling and at least one Agrobacterium strain that harbors a vector carrying a desired polynucleotide; (b) (i) producing callus from said transformed seedling; (iii) inducing shoot and root formation from said callus to produce a plantlet; (c) growing said plantlets into plants; and (d) screening said plants to determine if said desired polynucleotide is incorporated into the genome of at least one cell of said plant, wherein a plant comprising said desired polynucleotide in said genome is a transgenic plant.
- 121. The method of claim 120, wherein said germinating plant seedling are from a monocotyledenous plant.
- 122. The method of claim 121, wherein said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, rice, oat, barley, sorghum, orchid, iris, lily, onion, sugarcane, sorghum.
- 123. The method of claim 122, wherein said turfgrass is selected from the group consisting of Agrostis spp., Poa pratensis, Lolium spp., Festuca arundinacea, Festuca rubra commutata, Cynodon dactylon, Pennisetum clandestinum, Stenotaphrum secundatum, Zoysia japonica, and Dichondra micrantha.
- 124. The method of claim 120, wherein said germinating plant seedling are from a dicotyledenous plant.
- 125. The method of claim 124, wherein said dicotyledenous plant is selected from the group consisting of cotton, tobacco, Arabidopsis, tomato, potato, sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, and cactus.
- 126. The method of claim 120, wherein expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genome, wherein said trait is selected from the group consisting of increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.
- 127. The method of claim 120, wherein said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5′-untranslated region of said gene, the 3′-untranslated region of said gene, the leader sequence associated with said gene, or the trailer sequence associated with said gene.
- 128. The method of claim 8 wherein said desired polynucleotide encodes a peptide or protein that is an antifungal, a nutritional peptide or protein, a transcription factor, a receptor that binds to pathogen-derived ligands, a hemoglobin, an oxidase, an enzyme of the lignin biosynthesis pathway, an enzyme of industrial value, or is an antigen.
- 129. The method of claim 120, wherein said desired polynucleotide is operably linked to a promoter and a terminator.
- 130. The method of claim 129, wherein the sequences of said promoter and said terminator naturally occur in the genome of plants.
- 131. The method of claim 120, wherein said vector comprises (a) a T-DNA or a P-DNA that comprises (i) said desired polynucleotide, and (ii) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants; and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.
- 132. The method of claim 131, wherein said backbone integration marker gene is operably linked to a promoter and a terminator.
- 133. The method of claim 132, wherein said backbone integration marker is a cytokinin gene.
- 134. The method of claim 133, wherein said cytokinin gene is IPT, and said plant is a dicotyledon plant.
- 135. The method of claim 131, wherein said backbone integration marker is PGA22, TZS, HOC1, CKI1, and ESR1.
- 136. The method of claim 120, wherein the step of screening comprises detecting the presence of said desired polynucleotide in cells of said transgenic plant.
- 137. The method of claim 120, further comprising producing progeny from said transgenic plant and detecting the presence of said desired polynucleotide in cells of said progeny.
- 138. The method of claim 131, wherein said border-like sequences of said P-DNA range in size from 20 to 100 bp and share between 52% and 96% sequence identity with a T-DNA border sequence from Agrobacterium tumafaciens.
- 139. The method of claim 131, wherein expression of said selectable marker gene confers fertilizer tolerance to said transgenic plants and progeny thereof.
- 140. The method of claim 139, wherein said selectable marker gene that confers fertilizer tolerance is a selectable marker gene that confers resistance to cyanamide.
- 141. The method of claim 140, wherein said selectable marker gene that confers resistance to cyanamide is selected from the group consisting of the CAH gene and homologs thereof, the Aspergillus CAH-H1 gene, a fungal gene comprising a sequence at least 70% homology to CAH-H1, and SEQ ID NO. 1.
- 142. The method of claim 131, wherein said selectable marker gene is operably linked to a yeast ADH terminator.
- 143. The method of claim 131, wherein said selectable marker gene is an antibiotic resistance gene.
- 144. The method of claim 143, wherein said antibiotic resistance gene is selected from the group consisting of hygromycin phosphotransferase, neomycin phosphotransferase, streptomycin phosphotransferase, and bleomycin-binding protein.
- 145. The method of claim 131, wherein said selectable marker gene is a herbicide resistance gene.
- 146. The method of claim 145, wherein said herbicide resistance gene is selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, and phosphinothricin acetyl transferase.
- 147. The method of claim 120, wherein said solution is vortexed from about 60 seconds to several hours.
- 148. The method of claim 120, wherein said solution is vortexed for about 5 minutes to about 30 minutes.
- 149. The method of claim 79, further comprising the step of growing the seedling of (e) into a plant, wherein said plant is a transformed plant and wherein at least one cell of said transformed plant comprises in its genome said desired polynucleotide.
- 150. The method of claim 149, further comprising crossing said transformed plant with a non-transformed plant to produce at least one progeny plant that comprises said desired polynucleotide in its genome.
- 151. The method of claim 149, further comprising selfing said transformed plant to produce at least one progeny plant that comprises said desired polynucleotide in its genome.
- 152. The method of claim 79, wherein said desired polynucleotide is operably linked to a promoter and a terminator.
- 153. The method of claim 79, wherein said desired polynucleotide consists essentially of a sequence that is native to said selected plant, native to a plant from the same species, or is native to a plant that is sexually interfertile with said selected plant.
- 154. The method of claim 152, wherein said desired polynucleotide, said promoter, and said terminator consist essentially of sequences that are endogenous to a sequence naturally found in a plant.
- 155. The method of claim 79, wherein the modification of expression of a functional gene results in the modification of a trait to plants that comprise said desired polynucleotide in their genomes, wherein said trait is selected from the group consisting of increased drought tolerance, reduced height, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.
- 156. The method of claim 84, wherein said first vector and said second vector are both present in the same strain of Agrobacterium.
- 157. The method of claim 156, wherein said first vector is present in a first strain of Agrobacterium and said second vector is present in a second, different strain of Agrobacterium.
- 158. The method of claim 56, wherein said plant tissue is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 159. The method of claim 158, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 160. The method of claim 56, wherein double-stranded breaks are induced in a genome in a cell of said plant tissue by exposing said plant tissue to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 161. A polynucleotide that has at least 90% sequence identity to any one of SEQ ID NOs. 1, 6, or 9.
- 162. A cyanamide tolerance gene comprising the sequence of SEQ ID NO. 1.
- 163. A rice actin-1 gene terminator sequence comprising the sequence of SEQ ID NO. 6.
- 164. A plant-like promoter, comprising the sequence of SEQ ID NO. 9.
- 165. The method of claim 9, wherein said antifungal peptide or protein is D4E1 or alfalfa AFP, wherein said transcription factor is CBF3, wherein said hemoglobin is selected from the group consisting of VhB, cytokines, starch associated R1, polyphenol oxidase, ADP-glucose pyrophosphorylase, wherein said oxidase is GA20 oxidase, GA2 oxidase.
- 166. The method of claim 12, wherein said selectable marker gene is a positive selection marker.
- 167. The method of claim 166, wherein said positive selection marker is phosphomannose isomerase or xylose isomerase.
- 168. The method of claim 99, wherein said antifungal peptide or protein is D4E1 or alfalfa AFP, wherein said transcription factor is CBF3, wherein said hemoglobin is selected from the group consisting of VhB, cytokines, starch associated R1, polyphenol oxidase, ADP-glucose pyrophosphorylase, wherein said oxidase is GA20 oxidase, GA2 oxidase.
- 169. The method of claim 128, wherein said antifungal peptide or protein is D4E1 or alfalfa AFP, wherein said transcription factor is CBF3, wherein said hemoglobin is selected from the group consisting of VhB, cytokines, starch associated R1, polyphenol oxidase, ADP-glucose pyrophosphorylase, wherein said oxidase is GA20 oxidase, GA2 oxidase.
- 170. The method of claim 55, wherein said antifungal peptide or protein is D4E1 or alfalfa AFP, wherein said transcription factor is CBF3, wherein said hemoglobin is selected from the group consisting of VhB, cytokines, starch associated R1, polyphenol oxidase, ADP-glucose pyrophosphorylase, wherein said oxidase is GA20 oxidase, GA2 oxidase.
- 171. The method of claim 77, wherein said antifungal peptide or protein is D4E1 or alfalfa AFP, wherein said transcription factor is CBF3, wherein said hemoglobin is selected from the group consisting of VhB, cytokines, starch associated R1, polyphenol oxidase, ADP-glucose pyrophosphorylase, wherein said oxidase is GA20 oxidase, GA2 oxidase.
- 172. The method of claim 37, wherein said selectable marker gene is a positive selection marker.
- 173. The method of claim 172, wherein said positive selection marker is phosphomannose isomerase or xylose isomerase.
- 174. The method of claim 56, wherein said selectable marker gene is a positive selection marker.
- 175. The method of claim 174, wherein said positive selection marker is phosphomannose isomerase or xylose isomerase.
- 176. The method of claim 102, wherein said selectable marker gene is a positive selection marker.
- 177. The method of claim 176, wherein said positive selection marker is phosphomannose isomerase or xylose isomerase.
- 178. The method of claim 131, wherein said selectable marker gene is a positive selection marker.
- 179. The method of claim 178, wherein said positive selection marker is phosphomannose isomerase or xylose isomerase.
- 180. The method of claim 1, wherein said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 181. The method of claim 180, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 182. The method of claim 1, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 183. The method of claim 79, wherein said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 184. The method of claim 183, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 185. The method of claim 79, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 186. The method of claim 88, wherein said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 187. The method of claim 186, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 188. The method of claim 88, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 189. The method of claim 91, wherein said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 190. The method of claim 189, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 191. The method of claim 91, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 192. The method of claim 120, wherein said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 193. The method of claim 192, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 194. The method of claim 120, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 195. A method for producing a transgenic plant, comprising transforming a germinating plant seedling, or explant thereof, with a desired polynucleotide, wherein the seedling is exposed to a chemical prior to, during, and/or after transformation with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue.
- 196. The method of claim 195, wherein said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.
- 197. The method of claim 195, wherein double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.
- 198. The method of claim 195, wherein the seedling is transformed by particle bombardment, polyethylene glycol treatment, liposomal nucleic acid compositions, microinjection, whiskers, electroporation, or sonication.
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a Non-Provisional of U.S. application Ser. Nos. 60/365,527, filed Mar. 20, 2002, and 60/377,597, filed May 06, 2002, which are both incorporated herein by reference in its entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60365527 |
Mar 2002 |
US |
|
60377597 |
May 2002 |
US |