Method of producing transgenic maize using direct transformation of commercially important genotypes

Abstract

Methods for transformation of maize with nucleic acid sequences of interest are disclosed. The method involves subjecting immature zygotic embryos or Type I callus to high velocity microprojectile bombardment. The method is capable of producing transformed maize lines of commercial importance and their hybrid combinations.

What is claimed is:  
     
         1 . A method for producing stably transformed fertile maize plants, said method comprising: 
 obtaining an immature embryo from a maize plant;    delivering a nucleic acid sequence of interest by microprojectile bombardment within 14 days to said immature embryo;    selecting for transformed cells; and,    regenerating fertile transformed slants.    
     
     
         2 . The method of  claim 1  wherein said maize plant is selected from the genotypes CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 and CG00689.  
     
     
         3 . The method of  claim 1  wherein an immature embryo is pretreated with an osmotically-active substance while in the presence of a nutrient medium.  
     
     
         4 . The method of  claim 3  wherein an osmotically-active substance is selected from sucrose, sorbitol, polyethylene glycol, glucose and mannitol.  
     
     
         5 . The method of  claim 1  wherein said nucleic acid sequence codes for an insecticidal protein.  
     
     
         6 . The method of  claim 5  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         7 . The method of  claim 6  wherein said nucleic acid sequence codes for the protein endotoxin of  Bacillus thurigiensis.    
     
     
         8 . The method of  claim 7  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         9 . The method of  claim 5  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         10 . The method of  claim 5  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         11 . The method of  claim 6  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         12 . The method of  claim 7  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         13 . The method of  claim 8  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         14 . The method of  claim 9  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase-promoter and a root-preferred promoter.  
     
     
         15 . The method of  claim 1  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.  
     
     
         16 . The method of  claim 15  whereby the transformed cells are selected visually using the expression of anthocyanin controlled by the introduced genes.  
     
     
         17 . A stably transformed maize plant produced by: 
 obtaining an immature embryo from a maize plant;    delivering a nucleic acid sequence of interest by microprojectile bombardment within 14 days to said immature embryo;    selecting for transformed cells; and,    regenerating fertile transformed plants.    
     
     
         18 . The stably transformed maize plant of  claim 17  having essentially one of the genotypes known as CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or CG00689.  
     
     
         19 . A hybrid obtained by crossing the stably transformed plant of  claim 18  with another maize plant.  
     
     
         20 . The maize plant of  claim 18  wherein said nucleic acid sequence encodes an insecticidal protein.  
     
     
         21 . The maize plant of  claim 20  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         22 . The maize plant of  claim 21  wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.  
     
     
         23 . The maize plant of  claim 18  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         24 . The maize plant of  claim 20  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         25 . The maize plant of  claim 20  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         26 . The maize plant of  claim 21  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         27 . The maize plant of  claim 22  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         28 . The maize plant of  claim 23  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         29 . The maize plant of  claim 24  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         30 . The maize plant of  claim 18  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.  
     
     
         31 . The hybrid of  claim 19  wherein said nucleic acid sequence encodes an insecticidal protein.  
     
     
         32 . The hybrid of  claim 31  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         33 . The hybrid of  claim 32  wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.  
     
     
         34 . The hybrid of  claim 31  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         35 . The hybrid of  claim 31  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         36 . The hybrid of  claim 31  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         37 . The hybrid of  claim 32  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         38 . The hybrid of  claim 33  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         39 . The hybrid of  claim 34  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         40 . The hybrid of  claim 35  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         41 . The hybrid of  claim 19  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.  
     
     
         42 . A method for producing stably transformed fertile maize plants, said method comprising: 
 obtaining Type I embryogenic callus derived from a viable portion of a maize plant;    delivering a nucleic acid sequence of interest by microprojectile bombardment to said Type I embryogenic callus;    selecting for transformed cells; and,    regenerating fertile transformed plants.    
     
     
         43 . The method of  claim 42  wherein said maize plant is selected from the genotypes CG00526, LH51, CG00708, LH82, G00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 and CG00689.  
     
     
         44 . The method of  claim 42  wherein a Type I embryogenic callus is pretreated with an osmotically-active substance while in the presence of a nutrient medium.  
     
     
         45 . The method of  claim 44  wherein an osmotically-active substance is selected from sucrose, sorbitol, polyethylene glycol, glucose, mannitol.  
     
     
         46 . The method of  claim 42  wherein said nucleic acid sequence codes for an insecticidal protein.  
     
     
         47 . The method of  claim 46  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         48 . The method of  claim 47  wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.  
     
     
         49 . The method of  claim 46  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         50 . The method of  claim 46  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         51 . The method of  claim 46  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         52 . The method of  claim 47  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         53 . The method of  claim 48  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         54 . The method of  claim 49  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         55 . The method of  claim 50  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         56 . The method of  claim 42  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.  
     
     
         57 . A stably transformed maize plant produced by: 
 obtaining Type I embryogenic callus derived from a viable portion of a maize plant;    delivering a nucleic acid sequence of interest by microprojectile bombardment to said Type I embryogenic callus;    selecting for transformed cells; and,    regenerating fertile transformed plants.    
     
     
         58 . The stably transformed maize plant of  claim 57  having essentially one of the genotypes known as CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or CG00689.  
     
     
         59 . A hybrid obtained by crossing the stably transformed plant of  claim 58  with another maize plant.  
     
     
         60 . The maize plant of  claim 58  wherein said nucleic acid sequence encodes an insecticidal protein.  
     
     
         61 . The maize plant of  claim 60  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         62 . The maize plant of  claim 61  wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.  
     
     
         63 . The maize plant of  claim 58  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         64 . The maize plant of  claim 60  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         65 . The maize plant of  claim 60  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         66 . The maize plant of  claim 61  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         67 . The maize plant of  claim 62  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         68 . The maize plant of  claim 63  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         69 . The maize plant of  claim 64  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         70 . The maize plant of  claim 58  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.  
     
     
         71 . The hybrid of  claim 59  wherein said nucleic acid sequence encodes an insecticidal protein.  
     
     
         72 . The hybrid of  claim 71  wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.  
     
     
         73 . The hybrid of  claim 72  wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.  
     
     
         74 . The hybrid of  claim 71  wherein said nucleic acid sequence is a maize optimized coding sequence.  
     
     
         75 . The hybrid of  claim 71  wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.  
     
     
         76 . The hybrid of  claim 71  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         77 . The hybrid of  claim 72  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         78 . The hybrid of  claim 73  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         79 . The hybrid of  claim 74  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         80 . The hybrid of  claim 75  wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.  
     
     
         81 . The hybrid of  claim 59  wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.