Non-transgenic herbicide resistant plants

Abstract

The present invention relates to the production of a non-transgenic plant resistant or tolerant to a herbicide of the phosphonomethylglycine family, e.g., glyphosate. The present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS). The mutated protein, which substantially maintains the catalytic activity of the wild-type protein, allows for increased resistance or tolerance of the plant to a herbicide of the phosphonomethylglycine family, and allows for the substantially normal growth or development of the plant, its organs, tissues or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide. The present invention also relates to a non-transgenic plant cell in which the EPSPS gene has been mutated, a non-transgenic plant regenerated therefrom, as well as a plant resulting from a cross using a regenerated non-transgenic plant having a mutated EPSPS gene.

1 . A non-transgenic herbicide resistant plant which comprises a plant that expresses a mutant EPSPS gene product instead of a wild-type EPSPS gene product wherein the wild-type EPSPS gene product has been mutated at one or more amino acid positions, said positions selected from the group consisting of Leu 173 , Ala 179 , Met 180 , Arg 181 , Ser 98 , Ser 255  and Leu 198  in  Arabidopsis  or at an analogous amino acid residue in an EPSPS paralog and said plant has substantially normal growth as compared to a plant expressing the wild-type EPSPS gene product.  
     
     
         2 . A non-transgenic herbicide resistant plant which comprises a plant that expresses a mutant EPSPS gene product instead of a wild-type EPSPS gene product wherein the wild-type EPSPS gene product has been mutated at one or more amino acid positions, said positions selected from the group consisting of Leu 173 , Ala 179 , Met 180 , Arg 181 , Ser 98 , Ser 255  and Leu 198  in  Arabidopsis  or at an analogous amino acid residue in an EPSPS paralog and said plant has substantially the same catalytic activity as compared to a plant expressing the wild-type EPSPS gene product.  
     
     
         3 . The plant according to  claim 1  in which the herbicide is a member of the phosphonomethylglycine family.  
     
     
         4 . The plant according to  claim 3  in which the member of the phosphonomethylglycine family is glyphosate.  
     
     
         5 . The plant according to  claim 1  in which the positions in the  Zea mays  paralog are selected from the group consisting of Leu 97 , Ala 103 , Met 104 , Arg 105 , Ser 23 , Ser 179  and Leu 122 .  
     
     
         6 . The plant according to  claim 1  in which the positions in the  Brassica napus  paralog are selected from the group consisting of Leu 169 , Ala 175 , Met 176 , Arg 177 , Ser 94 , Ser 251  and Leu 194 .  
     
     
         7 . The plant according to  claim 1 , in which the positions in the  Petunia hybrida  are selected from the group consisting of Leu 169 , Ala 175 , Met 176 , Arg 177 , Ser 94 , Ser 251  and Leu 194 .  
     
     
         8 . The plant according to  claim 1  in which the plant is selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugar beet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus, apple, lettuce, peas, lentils, grape and turf grasses.  
     
     
         9 . The plant according to  claim 1  in which the mutated gene results in one or more of the following amino acid substitutions in the EPSPS enzyme in comparison with the wild-type sequence: 
 (i) Leu 173 -Phe    (ii) Ala 179 -Gly    (iii) Met 180 -Cys    (iv) Arg 181 -Leu or Ser    (v) Ser 98 -Asp    (vi) Ser 255 -Ala    (vii) Leu 198 -Lys.    
     
     
         10 . The plant according to  claim 6  in which the mutated gene results in one or more of the following amino acid substitutions in the EPSPS enzyme in comparison with the wild-type sequence: 
 (i) Leu 97 -Phe    (ii) Ala 103 -Gly    (iii) Met 104 -Cys    (iv) Arg 105 -Leu or Ser    (v) Ser 23 -Asp    (vi) Ser 179 -Ala    (vii) Leu 122 -Lys.    
     
     
         11 . The plant according to  claim 7  in which the mutated gene results in one or more of the following amino acid substitutions in the EPSPS enzyme in comparison with the wild-type sequence: 
 (i) Leu 169 -Phe    (ii) Ala 175 -Gly    (iii) Met 176 -Cys    (iv) Arg 177 -Leu or Ser    (v) Ser 94 -Asp    (vi) Ser 251 -Ala    (vii) Leu 194 -Lys.    
     
     
         12 . The plant according to  claim 8  in which the mutated gene results in one or more of the following amino acid substitutions in the EPSPS enzyme in comparison with the wild-type sequence: 
 (i) Leu 169 -Phe    (ii) Ala 175 -Gly    (iii) Met 176 -Cys    (iv) Arg 177 -Leu or Ser    (v) Ser 94 -Asp    (vi) Ser 251 -Ala    (vii) Leu 194 -Lys.    
     
     
         13 . An isolated mutant EPSPS protein comprising the amino acid sequence depicted in SEQ ID NO:2, in which any one or more of the following amino acid positions are changed: Leu 173  is replaced with Phe, Ala 179  is replaced with Gly, Met 180  is replaced with Cys, Arg 181  is replaced with Leu or Ser, Ser 98  is replaced with Asp, Ser 255  is replaced with Ala and Leu 198  is replaced with Lys, which mutant EPSPS protein has increased resistance or tolerance to a herbicide, which herbicide is a member of the phosphonomethylglycine family.