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 independant transgene loci, where the selectable marker genes or transgenic elements can be segregated in the progeny.

1 - 65 . (canceled) 
     
     
         66 . A DNA construct comprising a T-DNA, wherein the T-DNA comprises a first plant expression cassette and a second plant expression cassette, wherein said first plant expression cassette comprises a conditional lethal gene under the control of a tissue specific promoter and said second plant expression cassette comprises a selectable marker gene or an agronomic gene of interest. 
     
     
         67 . The DNA construct of  claim 66 , wherein said tissue specific promoter is a plant male reproductive tissue specific promoter or a plant pollen specific promoter and provides expression of said conditional lethal gene in the male tissues or pollen of a plant. 
     
     
         68 . The DNA construct of  claim 66 , further comprising a second T-DNA comprising a third plant expression cassette, wherein said third plant expression cassette comprises a selectable marker gene or agronomic gene of interest. 
     
     
         69 . The DNA construct of  claim 68 , wherein said second T-DNA further comprises a fourth plant expression cassette, wherein said fourth plant expression cassette comprises a second conditional lethal gene heterologous to the conditional lethal gene contained in the first plant expression cassette. 
     
     
         70 . The DNA construct of  claim 66 , wherein said conditional lethal gene is selected from the group consisting of a phosphonate monoester hydrolase, a glyphosate oxidoreductase, a carboxy ester hydrolase, an N-acetyl-L-ornithine deacetylase, a P450 monooxygenase, a viral thymidine kinase, a cytosine deaminase, an indoleacetamide hydrolase and β-glucuronidase. 
     
     
         71 . The DNA construct of  claim 66 , further comprising one or more plasmid maintenance elements required for propagation of said DNA construct in bacteria. 
     
     
         72 . The DNA construct of  claim 71 , wherein said plasmid maintenance elements comprise at least one origin of replication and an antibiotic marker gene. 
     
     
         73 . The DNA construct of  claim 66 , wherein said selectable marker gene is an antibiotic selectable marker gene that provides tolerance to an antibiotic selected from the group consisting of hygromycin, kanamycin, bleomycin, G418, streptomycin and spectinomycin, or an herbicide selectable marker gene that provides tolerance to an herbicide selected from the group consisting of glyphosate, glufosinate, sulfonylureas, imidazol nones, bromoxynil, delapon, cyclohezanedione, a protoporphyrinogen oxidase inhibitor, and isoxaslutole herbicides. 
     
     
         74 . The DNA construct of  claim 66 , wherein said agronomic gene of interest provides an agronomic trait to a plant following transformation of said agronomic gene of interest into the plant, said agronomic trait comprising: herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance, mycoplasma disease resistance, modified oil 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, improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production. 
     
     
         75 . A method of controlling the dissemination of transgenes by pollen, said method comprising the steps of:
 a) transforming a plant cell with a DNA construct comprising a T-DNA, said T-DNA comprising a first plant expression cassette and a second plant expression cassette, wherein said first plant expression cassette comprises a selectable marker gene or an agronomic gene of interest and said second plant expression cassette comprises a conditional lethal gene under the control of a plant pollen specific promoter or a male reproductive tissue specific promoter; and   b) growing said plant cell into a first parent plant comprising said T-DNA.   
     
     
         76 . The method of  claim 75 , further comprising the step of:
 c) applying an effective amount of a protoxin to the first parent plant, wherein the protoxin is converted into a toxin by said conditional lethal gene, thereby causing impairment or death of pollen of the first parent plant expressing said conditional lethal gene.   
     
     
         77 . The method of  claim 75 , further comprising the steps of:
 c) crossing said first parent plant with a second parent plant to produce a hybrid plant; and   d) harvesting seed from said hybrid plant.   
     
     
         78 . The method of  claim 77 , further comprising the steps of:
 e) planting said seed from said hybrid plant in a field under conditions that cause the germination of said seed and growth into a hybrid plant; and   f) treating the hybrid plant with an effective amount of a protoxin, wherein the protoxin is converted into a toxin by said conditional lethal gene, thereby causing impairment or death of pollen expressing said conditional lethal gene.   
     
     
         79 . The method of  claim 78 , wherein said protoxin comprises a phosphonate ester of glyphosate, a carboxylate ester of glyphosate, glyphosate, a N-acetyl-L-phosphinothricin, sulfonamide R7402 protoxin, or a glucuronoside conjugate of an herbicide. 
     
     
         80 . The method of  claim 75 , wherein said DNA construct further comprises a second T-DNA comprising a third plant expression cassette, said third plant expression cassette comprising a selectable marker gene or agronomic gene of interest. 
     
     
         81 . The method of  claim 80 , wherein said second T-DNA further comprises a fourth plant expression cassette, wherein said fourth plant expression cassette comprises a second conditional lethal gene heterologous to the conditional lethal gene contained in the second plant expression cassette. 
     
     
         82 . The method of  claim 75 , wherein said conditional lethal gene is selected from the group consisting of a phosphonate monoester hydrolase, a glyphosate oxidoreductase, a carboxy ester hydrolase, an N-acetyl-L-ornithine deacetylase, a P450 monooxygenase, a viral thymidine kinase, a cytosine deaminase, an indoleacetamide hydrolase and a β-glucuronidase. 
     
     
         83 . The method of  claim 75 , wherein said selectable marker gene is an antibiotic selectable marker gene that provides tolerance to an antibiotic selected from the group consisting of hygromycin, kanamycin, bleomycin, G418, streptomycin and spectinomycin, or an herbicide selectable marker gene that provides tolerance to an herbicide selected from the group consisting of glyphosate, glufosinate, sulfonylureas, imidazol nones, bromoxynil, delapon, cyclohezanedione, a protoporphyrinogen oxidase inhibitor, and isoxaslutole herbicides. 
     
     
         84 . The method of  claim 75 , wherein said selectable marker gene encodes an enzyme selected from the group consisting of a class II EPSPS inhibitor resistant enzyme, a class I EPSPS inhibitor resistant enzyme, an aceto-lactone synthase inhibitor resistant enzyme, and a glutamine synthase inhibitor enzyme. 
     
     
         85 . The method of  claim 75 , wherein said agronomic gene of interest provides an agronomic trait to a plant following transformation of said agronomic gene of interest into the plant, said agronomic trait comprising: herbicide tolerance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance, mycoplasma disease resistance, modified oil 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, improved flavor, nitrogen fixation, hybrid seed production, reduced allergenicity, biopolymers, or biofuel production.