Methods and compositions for obtaining marker-free transgenic plants
AssigneeMonsanto Technology LLC
InventorsXudong Ye, Larry A. Gilbertson, Shihshieh Huang, Susan J. Johnson, Michael W. Petersen, David Walters, Paul S. Chomet
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Abstract
The invention provides methods and compositions for identifying transgenic seed that contain a transgene of interest, but lack a marker gene. Use of an identification sequence that results in a detectable phenotype increases the efficiency of screening for seed and plants in which transgene sequences not linked to a gene of interest have segregated from the sequence encoding a gene of interest.
Claims40
A transgenic cell transformed with and comprising each of:
- (1) a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants;
- and (2) a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a seed operably linked to a DNA cassette that confers a detectable phenotype at least in seeds of a plant comprising the DNA cassette;
- and a marker gene operably linked to a promoter functional in plants;
- wherein the detectable phenotype is a tissue ablation phenotype.
A composition comprising a mixture of (1) a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants;
- and (2) a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a seed operably linked to a DNA cassette that confers a detectable phenotype at least in seeds of a plant comprising the DNA cassette;
- and a marker gene operably linked to a promoter functional in plants wherein the detectable phenotype is selected from the group consisting of:
- altered seed size, change in seed shape, change in seed opacity, change in seed color, change in seed texture, change in seed germinability, nonviable pollen, a defective seed, an aborted seed, and change in seed viability;
- and wherein the detectable phenotype is not conferred by gus, gfp, or lux.
A composition comprising bacterial cells, the composition comprising (1) a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants;
- and (2) a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a seed operably linked to a DNA cassette that confers a detectable phenotype at least in seeds of a plant comprising the DNA cassette;
- and a marker gene operably linked to a promoter functional in plants wherein the detectable phenotype is selected from the group consisting of:
- altered seed size, change in seed shape, change in seed opacity, change in seed color, change in seed texture, change in seed germinability, nonviable pollen, a defective seed, an aborted seed, and change in seed viability;
- and wherein the detectable phenotype is not conferred by gus, gfp, or lux.
A method of preparing marker-free seeds from a transgenic plant comprising the steps of:
- a) obtaining seeds from a transgenic plant, said plant comprising a first DNA segment comprising a nucleic acid of interest and a second DNA segment comprising a marker gene physically linked to a DNA cassette conferring a detectable phenotype at least in seeds of a plant, wherein said DNA cassette is operably linked to a promoter functional in said seed, wherein the detectable phenotype is a tissue ablation phenotype;
- b) screening the seeds for the absence of the detectable phenotype;
- and
- c) selecting at least a first seed that lacks the detectable phenotype to obtain a seed free of the marker gene.
Dependencies are inferred from the claim text.