Homologous recombination-mediated transgene deletion in plant cells

Abstract

A process to prepare a recombined transgenic Zea mays plant or plant cell from a first transgenic Zea mays plant cell, wherein the transgene in the recombinant plant or plant cell has an altered genetic structure relative to the genetic structure of the transgene in the first transgenic plant cell, due to homologous recombination-mediated transgene deletion.

1 . A method of preparing a fertile transgenic cell having an altered transgene insertion comprising: 
 a) obtaining a first transgenic cell, wherein the transgene insertion DNA sequence comprises an ancillary DNA sequence flanked by directly repeated DNA sequences;    b) obtaining a plurality of progeny cells of any generation of the first transgenic cell;    c) selecting a progeny transgenic in the presence of a negative selection agent wherein said ancillary DNA sequence flanked by directly repeated DNA sequences is deleted in the transgene insertion in the progeny transgenic cell as compared to the transgene insertion in the first transgenic cell.    
     
     
         2 . The method of  claim 1  wherein said directly repeated DNA sequences are not recognized by a site-specific recombinase.  
     
     
         3 . The method of  claim 1  wherein said transgenic cell is homozygous for the transgene insertion DNA sequence.  
     
     
         4 . The method of  claim 1  wherein the ancillary DNA sequence flanked by directly repeated DNA sequence comprises a selectable marker gene or a reporter gene.  
     
     
         5 . The method of  claim 4  wherein the ancillary DNA sequence flanked by directly repeated DNA sequence further comprises a negative selectable marker gene.  
     
     
         6 . The method of  claim 5  wherein said negative selectable marker gene comprises a pehA gene, a cytosine deaminase gene, a T-DNA gene 2 gene or a thymidine kinase gene.  
     
     
         7 . The method of  claim 6  wherein said negative selectable marker gene is a pehA gene.  
     
     
         8 . The method of  claim 4  wherein the ancillary DNA sequence flanked by directly repeated DNA sequence comprises a bar, nptII, EPSPS, CP4, pat, GFP, uidA, or cryIA(b) gene.  
     
     
         9 . The method of  claim 8  wherein said selectable marker gene is an NPTII, bar or pat gene.  
     
     
         10 . The method of  claim 1  wherein said transgenic cell is a plant callus cell.  
     
     
         11 . The method of  claim 10  wherein said plant callus cell is monocotyledonous.  
     
     
         12 . The monocotyledenous plant callus cell of  claim 1  wherein said callus cell is obtained from maize.  
     
     
         13 . The method of  claim 10  further comprising regenerating a transgenic plant from said plant callus cell wherein said ancillary DNA sequence flanked by directly repeated DNA sequence is deleted.  
     
     
         14 . The transgenic plant produced by the method of  claim 13  wherein said regenerated transgenic plant lacks said ancillary DNA sequence.  
     
     
         15 . The transgenic plant of  claim 14  wherein said plant lacks marker or reporter genes.  
     
     
         16 . A transgenic progeny plant of any generation of the transgenic plant of  claim 14 .  
     
     
         17 . A seed of the transgenic plant of  claim 14 .  
     
     
         18 . A transgenic progeny plant of any generation of the transgenic plant of  claim 15 .  
     
     
         19 . A seed of the transgenic plant of  claim 17 .  
     
     
         20 . The transgenic plant of  claim 14  wherein said plant is monocotyledonous.  
     
     
         21 . The transgenic plant of  claim 20  wherein said plant is maize.  
     
     
         22 . The transgenic plant of  claim 21  which is inbred.  
     
     
         23 . The transgenic plant of  claim 21  which is hybrid.