Nuclear male sterile plants, methods of producing same and methods to restore fertility

Abstract

The present invention is directed to the production of male sterile plants by providing them with a recombinant DNA capable of specific expression in the male reproductive system of a plant of the enzyme trehalose phosphate phosphatase (TPP). Restoration of the fertility can be established either by providing said male sterile plants with a recombinant DNA capable of expression of trehalose phosphate synthase (TPS) under control of an inducible promoter or with a recombinant DNA capable of expression of a suppressor protein which suppresses expression of TPP under control of an inducible promoter. This inducible restoration possibilities enable the maintenance site-specific recombination system is provided, by inserting two site-specific recombination sites flanking the recombinant DNA coding for TPP and crossing the male sterile lines with lines expressing the corresponding recombinase. By crossing the recombinase will excise the gene coding for TPP and fertile hybrids are produced.

1 . A method to make plants male sterile by transforming it with a recombinant DNA capable of expression of a protein in the tapetum, pollen and/or anthers, characterized in that the protein is trehalose phosphate phosphatase (TPP).  
     
     
         2 . A method according to  claim 1 , characterized in that the recombinant DNA comprising the gene coding for TPP is of bacterial, fungal, animal, plant or human origin, preferably derived from  Escherichia coli.    
     
     
         3 . Recombinant DNA comprising a tapetum, pollen and/or anther specific promoter and a gene coding for TPP, preferably a gene of bacterial, fungal, animal, plant or human origin, more preferably derived from  Escherichia coli.    
     
     
         4 . Vector comprising the recombinant DNA according to  claim 3 .  
     
     
         5 . Agrobacterium strain comprising the vector according to  claim 4 .  
     
     
         6 . Plant transformed with the Agrobacterium strain of  claim 5 .  
     
     
         7 . Male sterile plant, characterized in that it comprises a recombinant DNA according to  claim 3 .  
     
     
         8 . Male sterile plant, characterized in that it is made according to the method of  claim 1  or  claim 2 .  
     
     
         9 . Method for making a plant male sterile and susceptible to restoration of male fertility by transforming it with a recombinant DNA capable of expression in the tapetum, pollen and/or anthers, characterized in that the recombinant DNA comprises a gene coding for trehalose phosphate phosphatase (TPP) and that the recombinant DNA coding for TPP is flanked by target sites of a site-specific recombinase.  
     
     
         10 . Method for restoration of fertility in a male sterile plant made according to the method of  claim 9 , characterized in that the recombinant DNA coding for TPP is removed by providing said plant either through transformation or through crossing with a recombinant DNA capable of expressing a site-specific recombinase.  
     
     
         11 . Method for making a plant male sterile and susceptible to restoration of male fertility by transforming it with a recombinant DNA capable of expression in the tapetum, pollen and/or anthers, characterized in that the recombinant DNA comprises a first gene coding for trehalose phosphate phosphatase (TPP) and a second gene coding for a molecule capable of neutralizing the effect of TPP, which second gene is under control of an inducible promoter.  
     
     
         12 . Method according to  claim 11 , characterized in that the second gene is coding for trehalose phosphate synthase (TPS).  
     
     
         13 . Method according to  claim 11 , characterized in that the second gene is encoding an antisense-TPP sequence.  
     
     
         14 . Method for restoration of fertility in a male sterile plant made according to any of the methods of claims  11 - 13 , characterized in that the second gene is expressed by induction of the inducible promoter.  
     
     
         15 . Method for making a plant male sterile and susceptible to restoration of male fertility by transforming it with a recombinant DNA capable of expression in the tapetum, pollen and/or anthers, characterized in that the recombinant DNA comprises a gene coding for trehalose phosphate phosphatase (TPP) of which the expression is controlled by a suppressor molecule encoded by a gene which is controlled by an inducible promoter.  
     
     
         16 . Method for restoration of fertility in a male sterile plant made according to the method of  claim 15 , characterized in that the suppressor is expressed by induction of the inducible promoter.  
     
     
         17 . Method according to any of claims  11 - 16 , characterized in that the inducible promoter is a chemically inducable promoter.  
     
     
         18 . Method for restoration of fertility in a plant made male sterile by expression of trehalose phophate phosphatase, characterized in that said male sterile plant is treated with gibberellic acid.  
     
     
         19 . Method according to  claim 18 , characterized in that the flower buds of the plants are sprayed with a solution of gibberellic acid.  
     
     
         20 . Method according to  claim 18  or  19 , characterized in that the gibberellic acid solution is comprising GA4 and/or GA7.  
     
     
         21 . Male sterile plant produced by the method of  claim 9 ,  11 ,  12 ,  13 , or  15 .  
     
     
         22 . Male fertile plant produced by the method of  claim 10 ,  14 ,  16 ,  18 ,  19  or  20 .  
     
     
         23 . Method for producing a male sterile homozygous line by crossing a male sterile plant produced by the method of  claim 11 ,  12  or  13  with a male fertile plant produced by the method of  claim 14 .  
     
     
         24 . Method for producing a male sterile homozygous line by crossing a male sterile plant produced by the method of claim IS with a male fertile plant produced by the method of  claim 16 .  
     
     
         25 . Method for producing a male sterile homozygous line by crossing a male sterile plant produced by the method of  claim 1  or  2  with a male fertile plant produced by the method of  claim 18 ,  19  or  20 .  
     
     
         26 . A male sterile homozygous line produced by the method of  claim 23 ,  24  or  25 .  
     
     
         27 . Method for the production of fertile hybrid plants by 
 a. transforming a plant with a recombinant DNA capable of expressing a site-specific recombinase; and    b. crossing said plant with a male sterile plant produced by the method of  claim 9 .    
     
     
         28 . Method for the production of fertile hybrid plants by 
 a. transforming a plant with a recombinant DNA capable of neutralizing the effect of TPP; and    b. crossing said plant with a male sterile plant produced by the method of  claim 1  or  claim 2 .    
     
     
         29 . Method according to  claim 28 , characterized in that the recombinant DNA capable of neutralizing the effect of TPP is selected from the group comprising of a recombinant DNA comprising a gene coding for TPS, a recombinant DNA comprising a gene coding for a suppressor which can bind to the transcription initiation site of the TPP gene and a recombinant DNA comprising an antisense TPP sequence.  
     
     
         30 . Fertile hybrid plants produced by a method according to any of claims  27 - 29 .