Methods for the production of plants resistant to HPPD herbicides

Abstract

Methods for making transgenic plants that are resistant to HPPD herbicides are presented. Polynucleotides other than those from Pseudomonas fluorescens that encode resistant HPPD enzymes are enclosed for use in the process of making transgenic plants that are tolerant to HPPD-inhibiting herbicides.

1 . A method of selecting a polynucleotide which encodes a triketone resistant HPPD enzyme comprising screening a population of HPPD enzyme encoding sequences and selecting as those which encode a triketone resistant HPPD enzyme those sequences which encode an enzyme which in comparison with a control HPPD enzyme is at least 2.5 fold more resistant to a Formula 1 triketone herbicide compared to a Formula 3 herbicide or is at least 2.5 fold more resistant to a Formula 2 triketone herbicide compared to a Formula 4 herbicide, wherein said control enzyme is selected so as to exhibit substantially the same selection of polynucleotides as is obtained when the control enzyme is derived from  Arabidopsis.    
     
     
         2 . The method according to  claim 1 , wherein the control HPPD is derived from a dicot. 
     
     
         3 . The method according to  claim 1 , wherein the resistance of HPPD enzymes to herbicides is determined by measuring the rate of dissociation of the enzyme/herbicide complex. 
     
     
         4 . The method according to  claim 1  wherein the HPPD enzyme encoded by the selected polynucleotide has a kcat/Km hydroxyphenylpyruvate value in the range from 0.1 to 5 s −1  μM −1  at pH 7.0 and 25° C. 
     
     
         5 . A method of producing plants which are transgenic for a inhibitor resistant HPPD enzyme comprising: (a) transforming plant material with a polynucleotide obtained by the method according to  claim 1 ; (b) regenerating said plant material into a morphologically normal fertile plant, wherein said plants are transgenic for a inhibitor resistant HPPD enzyme. 
     
     
         9 . The method of  claim 1 , wherein the triketone resistant HPPD enzyme encoding sequences encode an enzyme which in comparison with a control HPPD enzyme is at least four fold more resistant to a Formula I triketone herbicide compared to a Formula 3 herbicide or is at least four fold more resistant to a Formula 2 triketone herbicide compared to a Formula 4 herbicide, wherein said control enzyme is selected so as to exhibit substantially the same selection of polynucleotides as is obtained when the control enzyme is derived from  Arabidopsis.    
     
     
         7 . The method according to  claim 2 , wherein said dicot is  Arabidopsis  or tobacco.