Methods and Compositions for the Selection of A Transgenic Plant

Abstract

Compositions and methods are provided to screen, identify, select, isolate, and/or regenerate targeted integration events using seed priming. Seed priming provides the identification of a seed having stably incorporated into its genome a site-specific recombinase mediated integration of a selectable marker at a target locus operably linked to a promoter active in the seed.

1 . A method to identify a plant having a DNA construct stably incorporated in its genome comprising:
 (a) providing a population of seeds, wherein at least one seed in the population has stably incorporated into its genome the DNA construct comprising the following operably linked components in the following order: a promoter active in the seed, a first recombination site, a polynucleotide encoding a first selection marker that confers resistance to a selective agent, and a second recombination site;   (b) contacting the population of seeds with a priming matrix comprising an effective concentration of the selection agent for a time sufficient to produce a population of primed seeds; and,   (c) incubating the population of primed seed under germination conditions to produce germinated seeds comprising the DNA construct,   
     thereby identifying plants having the DNA construct stably incorporated into their genome. 
   
   
       2 . The method of  claim 1 , wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other. 
   
   
       3 . The method of  claim 1 , wherein the selective agent comprises an herbicide, a growth regulator, or an antibiotic. 
   
   
       4 . The method of  claim 3 , wherein the selective agent is selected from the group consisting of glufosinate, phosphinothricin, glyphosate, bromoxynil, methotrexate, imidazolinones, sulfonylureas, cyanamide, kanamycin, G418, neomycin, and hygromycin B. 
   
   
       5 . The method of  claim 3 , wherein the first selection marker is selected from the group consisting of Bar, phosphinothricin acetyltransferase (PAT), glyphosate oxidoreductase (GOX), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), CP4, glyphosate N-acetyltransferase (GAT), bromoxynil nitrilase (BXN), dihydrofolate reductase, acetolactate synthase (ALS), cyanamide hydratase (CAH), neomycin phosphotransferase (nptII), aminoglycoside 3′-phosphotransferase (APH3′ II), and hygromycin phosphotransferase (hph). 
   
   
       6 . The method of  claim 5 , wherein the polynucleotide encoding the first selection marker has been synthesized using maize preferred codons. 
   
   
       7 . The method of  claim 1 , wherein the DNA construct comprises in the following order: a first expression unit comprising the promoter active in the seed operably linked to the first recombination site operably linked to the polynucleotide encoding the first selection marker; and a second expression unit comprising in the following order, a second promoter active in the plant operably linked to a polynucleotide of interest, and the second recombination site. 
   
   
       8 . The method of  claim 7 , wherein the polynucleotide of interest encodes a second selection marker, wherein the second selection marker is distinct from the first selection marker by having a different selection means. 
   
   
       9 . The method of  claim 1 , wherein the plant further has stably incorporated into its genome a polynucleotide encoding a site specific recombinase. 
   
   
       10 . The method of  claim 9 , wherein the site specific recombinase comprises a FLP recombinase, a Cre recombinase, a lambda integrase, a SSV1 integrase, or a phiC31 integrase. 
   
   
       11 . The method of  claim 1 , wherein at least one of the first or the second recombination sites are selected from the group consisting of a lox site, a mutant lox site, a FRT site, a mutant FRT site, an att site, and a mutant att site. 
   
   
       12 . The method of  claim 1 , wherein the plant is maize, wheat, rice, sorghum, rye, oat, barley, millet,  Brassica , alfalfa, sunflower, safflower, soybean, tobacco, cotton, or  Arabidopsis.    
   
   
       13 . A method of priming a seed comprising
 (a) providing a seed having stably incorporated into its genome a DNA construct comprising, in the following order, a promoter active in the seed operably linked to a first recombination site operably linked to a polynucleotide encoding a selection marker that confers resistance to a selective agent; and   (b) contacting the seed with a priming matrix comprising an effective concentration of the selective agent for a time sufficient to produce a primed seed.   
   
   
       14 . The method of  claim 13 , wherein the DNA construct comprises in the following order the promoter operably linked to the first recombination site operably linked the polynucleotide encoding the selection marker, and a second recombination site. 
   
   
       15 . The method of  claim 14 , wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other. 
   
   
       16 . A composition comprising a seed having stably incorporated into its genome a DNA construct comprising, in the following order, a promoter active in the seed operably linked to a first recombination site operably linked to a polynucleotide encoding a selection marker that confers resistance to a selective agent; and, a priming matrix comprising an effective concentration of the selective agent. 
   
   
       17 . The composition of  claim 16 , wherein the DNA construct comprises in the following order, the promoter operably linked to the first recombination site operably linked to the polynucleotide encoding the selection marker, and a second recombination site. 
   
   
       18 . The composition of  claim 17 , wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other. 
   
   
       19 . The composition  claim 16 , wherein the DNA construct further comprises a polynucleotide of interest. 
   
   
       20 . The composition of any one of  claim 16 , wherein the DNA construct further comprises a second selection marker. 
   
   
       21 . The composition of  claim 20 , wherein the second selection marker confers resistance to a second selective agent. 
   
   
       22 . The composition of  claim 20 , wherein the priming matrix further comprises the second selective agent. 
   
   
       23 . The composition of  claim 16 , wherein the seed is from a plant selected from the group consisting of maize, wheat, rice, sorghum, rye, oat, barley, millet,  Brassica , alfalfa, sunflower, safflower, soybean, tobacco, cotton, and  Arabidopsis.