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US 9,540,700 B2Granted
Methods and compositions for obtaining marker-free transgenic plants
AssigneeMonsanto Technology LLC
InventorsXudong Ye, Michael W. Petersen, Larry A. Gilbertson, David Walters, Susan J. Johnson, Shihshieh Huang, Paul S. Chomet
Life of this patent
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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.
Claims47
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 that is operably linked to a promoter functional in said seed,
- wherein the DNA cassette confers a detectable phenotype selected from the group consisting of:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- 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;
- wherein said detectable phenotype is not conferred by gus or gfp.
5 more elements
A DNA construct comprising:
- (a) 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
- (b) 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 in seeds that comprise 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:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- and
- wherein said detectable phenotype is not conferred by gus or gfp.
5 more elements
A transgenic plant co-transformed with a DNA construct, said DNA construct comprising:
- (a) 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
- (b) a second DNA construct containing 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 in seeds that comprise 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:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- and
- wherein said detectable phenotype is not conferred by gus or gfp.
5 more elements
A DNA construct comprising right and left T-DNA borders,
- wherein a first DNA segment, comprising a gene of interest operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from a right border;
- and wherein a second DNA segment comprising:
- a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette, and a marker gene operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from a second right border or a left border;
- wherein the detectable phenotype is selected from the group consisting of:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- and
- wherein said detectable phenotype is not conferred by gus or gfp.
4 more elements
A DNA construct comprising right and left T-DNA borders,
- wherein a first DNA segment comprising:
- a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette, and a marker gene operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from a right border;
- and wherein a second DNA segment, comprising a gene of interest operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from a left border;
- wherein the detectable phenotype is selected from the group consisting of:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- and
- wherein said detectable phenotype is not conferred by gus or gfp.
4 more elements
A DNA construct comprising in the 5′ to 3′ direction first and second right T-DNA borders,
- wherein a first DNA segment, comprising a gene of interest operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from the first right border;
- and wherein a second DNA segment comprising:
- a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette, and a marker gene operably linked to a promoter functional in plants, is located downstream in the 5′ to 3′ direction from the second right border;
- wherein the detectable phenotype is selected from the group consisting of:
- change in seed color, change in seed texture, change in seed shape, and change in seed opacity;
- and
- wherein said detectable phenotype is not conferred by gus or gfp.
4 more elements
Dependencies are inferred from the claim text.