Methods For Enhancing Segregation of Transgenes in Plants and Compositions Thereof

Abstract

The compositions and methods are provided that enhance the selection of transgenic plants having two T-DNA molecules integrated into a plant genome at different physical and genetic loci. The compositions are DNA constructs that comprise novel arrangements of T-DNA molecules containing genes of interest, positive selectable marker genes, and conditional lethal genes. The methods disclosed herein comprises transforming a plant cell to comprise the DNA constructs of the present invention, regenerating the plant cell into a plant and identifying independent transgene loci, where the selectable marker genes or transgenic elements can be segregated in the progeny.

1 . A DNA construct comprising at least two T-DNA molecules and at least two DNA segments separating said T-DNA molecules, wherein a conditional lethal gene resides in at least one DNA segment. 
     
     
         2 . A DNA construct of  claim 1 , wherein at least one conditional lethal gene resides in each DNA segment. 
     
     
         3 . A DNA construct of  claim 1 , wherein a conditional lethal gene encodes for a protein comprising: phosphonate monoester hydrolase, carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1 or β-glucuronidase. 
     
     
         4 . A DNA construct of  claim 1 , wherein the T-DNA molecules comprise plant expression cassettes providing agronomic traits selected from the group consisting of comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         5 . A DNA construct comprising at least two T-DNA molecules and at least two DNA segments separating said T-DNA molecules, wherein at least T-DNA molecule contains a selectable marker gene and a conditional lethal gene. 
     
     
         6 . A DNA construct of  claim 5 , wherein at least one DNA segment contains a conditional lethal gene that is heterologous to the conditional lethal gene contained in the T-DNA. 
     
     
         7 . A DNA construct comprising a first T-DNA and a second T-DNA, wherein the first T-DNA contains at least a first plant expression cassette and a second plant expression cassette, wherein said first plant expression cassette comprises a first conditional lethal gene under the control of a tissue specific promoter and said second plant expression cassette comprises a selectable marker gene or agronomic gene of interest, and said second T-DNA contains at least a third plant expression cassette and a fourth plant expression cassette, wherein said third plant expression cassette comprises a second conditional lethal gene heterologous to said first conditional lethal gene under the control of a tissue specific promoter and said fourth plant expression cassette comprises a selectable marker gene or agronomic gene of interest. 
     
     
         8 . A DNA construct of  claim 7 , wherein said conditional lethal gene encodes for a protein comprising: phosphonate monoester hydrolase, carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1 or β-glucuronidase. 
     
     
         9 . A DNA construct of  claim 7 , wherein said tissue specific promoter provides enhanced expression in male tissues or pollen. 
     
     
         10 . A method of the invention described herein for producing transgenic plant cells and plants comprising the steps of:
 a) introducing a DNA construct into a plant cell, wherein said DNA construct comprises a DNA molecule comprising a first T-DNA, and a DNA molecule comprising a second T-DNA, and a plant expression cassette that expresses a conditional lethal gene product is located in a position between said first T-DNA and said second T-DNA; and   b) treating said plant cell with a protoxin in amounts sufficient to cause the impairment of the cells when said protoxin is converted to a toxic compound by the expression product of the conditional lethal gene; and   c) regenerating unimpaired plant cells into fertile plants.   
     
     
         11 . A method of  claim 10 , wherein the conditional lethal gene product encodes for a protein comprising: phosphonate monoester hydrolase, carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1 or β-glucuronidase. 
     
     
         12 . A method of  claim 10 , wherein said protoxin comprises: phosphonate ester of glyphosate, carboxylate ester of glyphosate, glyphosate, N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin, or glucuronoside conjugate of a herbicide compound. 
     
     
         13 . A method of  claim 10 , wherein said DNA construct is introduced into a plant cell by an  Agrobacterium  mediated transformation method. 
     
     
         14 . A method of  claim 10 , wherein said first T-DNA and said second T-DNA comprise plant expression cassettes providing agronomic traits selected from the group consisting of comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         15 . A method of  claim 10 , wherein said treated plant cells that convert the protoxin into a toxic compound by the expression product of the conditional lethal gene are killed. 
     
     
         16 . A method of the invention described herein for producing transgenic plant cells and plants comprising the steps of:
 a) introducing a DNA construct into a plant cell, wherein said DNA construct comprises a DNA molecule comprising a first T-DNA, and a DNA molecule comprising a second T-DNA, and a plant expression cassette that expresses a conditional lethal gene product is located in a position between said first T-DNA and said second T-DNA; and   b) growing said plant cell into a fertile plant; and   c) collecting seeds from said fertile plant; and   d) planting the seeds to cause germination of the seeds and growth of the seedlings; and e) treating the seedlings with a protoxin in amounts sufficient to cause the impairment of cells of the treated seedlings when said protoxin is converted to a toxic compound by the expression product of the conditional lethal gene.   
     
     
         17 . A method of  claim 16 , wherein the conditional lethal gene product encodes for a protein comprising: phosphonate monoester hydrolase, carboxylate ester hydrolase, glyphosate oxidase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1 or β-glucuronidase. 
     
     
         18 . A method of  claim 16 , wherein said protoxin comprises: phosphonate ester of glyphosate, carboxylate ester of glyphosate, glyphosate, N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin, or glucuronoside conjugate of a herbicide compound. 
     
     
         19 . A method of  claim 16 , wherein said DNA construct is introduced into a plant cell by an  Agrobacterium  mediated transformation method. 
     
     
         20 . A method of  claim 16 , wherein said first T-DNA and said second T-DNA comprise plant expression cassettes providing agronomic traits selected from the group consisting of comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         21 . A method of  claim 16 , wherein said treated seedlings that convert the protoxin into a toxic compound by the expression product of the conditional lethal gene are killed. 
     
     
         22 . A method for stacking transgene traits in plants comprising the steps of:
 a) constructing a first DNA construct comprising a first T-DNA, wherein said first T-DNA provides a first plant expression cassette of at least one agronomic gene of interest, and a second T-DNA wherein said second T-DNA provides a second plant expression cassette of at least one positive selectable marker gene product functional in plant cells and a third plant expression cassette that provides a conditional lethal gene product functional in plant cells, wherein said second and third plant expression cassettes are in a tandem orientation and adjacent to each other in said second T-DNA; and   b) transforming said DNA construct into a plant cell; and   c) treating said plant cell with an effective amount of a positive selection compound for which tolerance is provided by said positive selectable marker gene product; and   d) regenerating a positive selection compound tolerant plant cell into a fertile plant; and   e) collecting seeds from said fertile plant; and   f) planting seeds from said fertile plant that germinate to produce seedling plants; and   g) treating the seedling plants with a protoxin in amounts sufficient to cause impairment to the plant cells of the treated seedlings when said protoxin is converted to a toxic compound by said conditional lethal gene product; and   h) selecting a seedling plant from the treated seedling plants for the presence of the first T-DNA and for no plant cell impairment caused by the conversion of the protoxin to a toxic compound; and   i) propagating said seedling plant selected in step (h) into a fertile plant; and   j) constructing a second DNA construct comprising a first T-DNA, wherein said first T-DNA provides a first plant expression cassette of at least one agronomic gene of interest different than said first DNA construct, and a second T-DNA, wherein said second T-DNA provides a second plant expression cassette of at least one positive selectable marker gene product functional in plant cells and a third plant expression cassette that provides a conditional lethal gene product functional in plant cells, wherein said second and third plant expression cassettes are in a tandem orientation and adjacent to each other in said second T-DNA; and   k) retransforming said fertile plant if step (i) with said second DNA construct; and   l) repeating steps c-g; and   m) selecting a seedling plant for the presence of the agronomic gene of interest of the first DNA construct and the agronomic gene of interest of the second DNA construct and for no plant cell impairment caused by the conversion of the protoxin to a toxic compound; and   n) propagating said seedling plant selected in step (m) into a fertile plant.   
     
     
         23 . A method of controlling the dissemination of transgenes by pollen comprising the steps of:
 a) constructing a DNA construct comprising a first T-DNA and a second T-DNA, said first T-DNA comprising a first plant expression cassette and a second plant expression cassette, wherein said first plant expression cassette provides a selectable gene product and said second plant expression cassette provides a conditional lethal gene product expressed under the control of a pollen specific promoter, and said second T-DNA segment comprising a third plant expression cassette and a fourth plant expression cassette, wherein said third plant expression cassette provides an agronomic gene of interest and said fourth plant expression cassette provides a conditional lethal gene product expressed under the control of a pollen promoter; and   b) transforming said DNA construct into a plant cell; and   c) growing said plant cell into a fertile first parent plant homozygous for said first and second T-DNA segments; and   d) crossing said fertile first parent plant with a fertile second parent plant to produce a hybrid plant; and   e) harvesting seed from said hybrid plant; and   f) planting said seed from said hybrid plant in a field under conditions that cause the germination of said seed and growth into plants; and   h) treating the plants of step (f) with an effective amount of a compound that is converted to a toxin by the conditional lethal gene product expressed under the control of a plant male tissue specific promoter to cause impairment of pollen expressing the conditional lethal gene product.   
     
     
         24 . A method of  claim 23 , wherein said conditional lethal gene product comprises phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, or β-glucuronidase 
     
     
         25 . A method of  claim 23 , wherein said compound that is converted to a toxin by the conditional lethal gene product comprises phosphonate ester of glyphosate, carboxylate ester of glyphosate, glyphosate, N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin, or glucuronoside conjugate of a herbicide. 
     
     
         26 . A method of  claim 23 , wherein said selectable gene product selected from the group consisting of class II EPSPS inhibitor resistant enzymes, class I EPSPS inhibitor resistant enzymes, aceto-lactone synthase inhibitor resistant enzymes, and glutamine synthase inhibitor enzymes. 
     
     
         27 . A method of  claim 23 , wherein said agronomic gene of interest provides an agronomic trait selected from the group consisting of comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         28 . A method of determining the expression pattern of a plant tissue specific DNA promoter for use in transgenic plants the steps comprising:
 b) constructing a two T-DNA construct comprising at least a first and a second plant expression cassette, wherein the first plant expression cassette has a constitutive promoter and resides in a first T-DNA, and the second plant expression cassette has a tissue specific promoter and resides in a second T-DNA; and   c) transforming the DNA construct into a plant cell; and   d) regenerating said plant cell into a fertile plant; and   e) assaying said plant for the presence of the plant expression cassettes; and   f) assaying said plant by a DNA detection method that identifies the linkage of the plant expression cassettes; and   g) assaying said plant for the expression pattern, or expression rate, or expression level of a gene product produced by the expression cassette having the tissue specific promoter; and   f) selecting said tissue specific promoter for use in the construction of additional DNA constructs.   
     
     
         29 . A method for providing a transgenic plant with independently segregating transgenes comprising the steps of:
 a) introducing a DNA molecule into a plant cell, wherein said DNA molecule comprises a DNA construct comprising a first and second DNA region, wherein said first DNA region comprises a first right border region, linked to at least one transgene of agronomic interest, linked to a first left border region, wherein said first left border region is linked to a second DNA region comprising a second left border region, linked to a positive selectable marker transgene, linked to at least one plasmid maintenance element, linked to a second right border region; and   b) regenerating said plant cell into a transgenic plant by positive selection provided by expression of said positive selectable marker transgene; and   c) selecting said transgenic plant for the presence of said transgene of agronomic interest; and   d) screening said transgenic plant by a DNA detection method that identifies the linkage of said transgene of agronomic interest and said positive selectable marker transgene; and   e) growing said transgenic plant into a fertile plant in which said transgene of agronomic interest and said positive selectable marker transgene are not linked.   
     
     
         30 . In the method of  claim 29 , further comprising the steps of:
 a) harvesting progeny from said fertile plant; and   b) selecting from said progeny those that contain said transgene of agronomic interest and do not contain said positive selectable marker transgene.   
     
     
         31 . A method for providing a transgenic plant with independently segregating transgenes comprising the steps of:
 a) introducing a DNA molecule into a plant cell, wherein said DNA molecule comprises a DNA construct comprising a first  Agrobacterium  Ti plasmid border region linked to first DNA region containing at least one transgene of agronomic interest, linked to a second  Agrobacterium  Ti plasmid border region, linked to a second DNA region containing a positive selectable marker transgene linked to at least one plasmid maintenance element, whereby the DNA molecule provides for the integration of one or both of the DNA regions into a plant genome; and   b) regenerating said plant cell into a transgenic plant by positive selection provided by expression of said positive selectable marker transgene; and   c) selecting said transgenic plant for the presence of said transgene of agronomic interest; and   d) screening said transgenic plant by a DNA detection method that identifies the linkage of said transgene of agronomic interest and said positive selectable marker transgene; and   e) growing said transgenic plant into a fertile plant.   
     
     
         32 . In the method of  claim 31 , further comprising the steps of:
 a) harvesting progeny from said fertile plant; and   b) selecting from said progeny those that contain said transgene of agronomic interest and do not contain said positive selectable marker transgene.   
     
     
         33 . In the method of  claim 32 , wherein said progeny are seeds or seedlings thereof. 
     
     
         34 . In the method of  claim 31 , wherein said transgene of agronomic interest provides an agronomic trait comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         35 . In the method of  claim 31 , wherein said transgenic plant is a crop plant. 
     
     
         36 . In the method of  claim 31 , wherein said positive selectable marker transgene provides tolerance to an antibiotic. 
     
     
         37 . In the method of  claim 31 , wherein said positive selectable marker transgene provides tolerance to a herbicide. 
     
     
         38 . In the method of  claim 37 , wherein said herbicide is selected from the group consisting of: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides. 
     
     
         39 . In the method of  claim 31 , wherein said first and second border region comprises an  Agrobacterium  Ti plasmid right border region or derived therefrom. 
     
     
         40 . In the method of  claim 31 , wherein said first and second border region comprises an  Agrobacterium  Ti plasmid left border region or derived therefrom. 
     
     
         41 . In the method of  claim 31 , wherein said first border region comprises an  Agrobacterium  Ti plasmid right border region or derived therefrom and said second border region comprises an  Agrobacterium  Ti plasmid left border region or derived therefrom 
     
     
         42 . In the method of  claim 31 , wherein said positive selectable marker transgene is linked to a conditional lethal transgene. 
     
     
         43 . In the method of  claim 42 , wherein said conditional lethal transgene is selected from the group consisting of: phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, and β-glucuronidase. 
     
     
         44 . In the method of  claim 31 , wherein said positive selectable marker transgene is linked to a plasmid maintenance element. 
     
     
         45 . A method for providing a transgenic plant with independently segregating transgenes comprising the steps of:
 a) introducing a DNA molecule into a plant cell, wherein said DNA molecule comprises a DNA construct comprising a first  Agrobacterium  Ti plasmid border region linked to first DNA region containing at least one transgene of agronomic interest, linked to a second  Agrobacterium  Ti plasmid border region, linked to a second DNA region containing a positive selectable marker transgene linked to at least one plasmid maintenance element, whereby the DNA molecule provides for the integration of one or both of the DNA regions into a plant genome; and   b) regenerating said plant cell into a transgenic plant by selection on an antibiotic or herbicide wherein tolerance to said antibiotic or herbicide is provided by expression of said positive selectable marker transgene; and   c) selecting said transgenic plant for the presence of said transgene of agronomic interest; and   c) growing said transgenic plant into a fertile plant that contains said transgene of agronomic interest and said positive selectable marker transgene; and   e) harvesting seeds from said fertile plant; and   f) selecting the seeds or seedlings thereof, that contain said transgene of agronomic interest and do not contain said positive selectable marker transgene.   
     
     
         46 . In the method of  claim 45 , wherein said transgene of agronomic interest provides an agronomic trait comprising herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance,  mycoplasma  disease resistance, modified oils production, high oil production, high protein production, germination and seedling growth control, enhanced animal and human nutrition, low raffinose, environmental stress tolerance, increased digestibility, industrial enzyme production, pharmaceutical peptides and small molecule production, improved processing traits, proteins improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, and biofuel production. 
     
     
         47 . In the method of  claim 45 , wherein said transgenic plant is a crop plant. 
     
     
         48 . In the method of  claim 45 , wherein said positive selectable marker transgene provides tolerance to an antibiotic. 
     
     
         49 . In the method of  claim 45 , wherein said positive marker transgene provides tolerance a herbicide. 
     
     
         50 . In the method of  claim 49 , wherein said herbicide is selected from the group consisting of glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides. 
     
     
         51 . In the method of  claim 45 , wherein said first and second border region comprises an  Agrobacterium  Ti plasmid right border region or derived therefrom. 
     
     
         52 . In the method of  claim 45 , wherein said first and second border region comprises an  Agrobacterium  Ti plasmid left border region or derived therefrom. 
     
     
         53 . In the method of  claim 45 , wherein said first border region is an  Agrobacterium  Ti plasmid right border region or derived therefrom and said second border region comprises an  Agrobacterium  Ti plasmid left border region or derived therefrom 
     
     
         54 . In the method of  claim 45 , wherein said positive selectable marker transgene is linked to a conditional lethal transgene. 
     
     
         55 . In the method of  claim 54 , wherein said conditional lethal transgene is selected from the group consisting of: phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, and β-glucuronidase. 
     
     
         56 . A DNA plasmid consisting of a first  Agrobacterium  Ti plasmid right border region linked to at least one transgene of agronomic interest linked to a second  Agrobacterium  Ti plasmid right border region linked to an antibiotic positive plant selectable marker transgene or a herbicide positive selectable marker transgene, linked to a plasmid maintenance element. 
     
     
         57 . A DNA plasmid of  claim 56 , wherein said antibiotic positive plant selectable marker transgene or herbicide selectable marker transgene is linked to a conditional lethal transgene. 
     
     
         58 . A DNA plasmid of  claim 57 , wherein said conditional lethal transgene is selected from the group consisting of: phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, β-glucuronidase. 
     
     
         59 . A DNA plasmid consisting of a first  Agrobacterium  Ti plasmid left border region linked to at least one transgene of agronomic interest linked to a second  Agrobacterium  Ti plasmid left border region linked to an antibiotic positive plant selectable marker transgene or a herbicide selectable marker transgene. 
     
     
         60 . A DNA plasmid of  claim 59 , wherein said antibiotic positive plant selectable marker transgene or herbicide selectable marker transgene is linked to a plasmid maintenance element. 
     
     
         61 . A DNA plasmid of  claim 59 , wherein said antibiotic positive plant selectable marker transgene or herbicide selectable marker transgene is linked to a conditional lethal transgene. 
     
     
         62 . A DNA plasmid of  claim 59 , wherein said conditional lethal transgene is selected from the group consisting of: phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, and β-glucuronidase. 
     
     
         63 . A DNA plasmid consisting of an  Agrobacterium  Ti plasmid right border region linked to at least one transgene of agronomic interest linked to an  Agrobacterium  Ti plasmid left border region linked to an antibiotic positive plant selectable marker transgene or a herbicide selectable marker transgene, linked to a plasmid maintenance element. 
     
     
         64 . A DNA plasmid of  claim 63 , wherein said antibiotic positive plant selectable marker transgene or herbicide selectable marker transgene is linked to a conditional lethal transgene. 
     
     
         65 . A DNA plasmid of  claim 64 , wherein said conditional lethal transgene is selected from the group consisting of: phosphonate monoester hydrolase, glyphosate oxidoreductase, carboxy ester hydrolase, N-acetyl-L-ornithine deacetylase, P450 monooxygenase CPY105A1, and β-glucuronidase.