Methods and compositions for producing male sterile plants

Abstract

Methods of making a targeted modification in a male fertility gene in the genome of a plant are disclosed. The methods involve contacting a plant cell with an engineered double-strand-break-inducing agent capable of inducing a double-strand break in a target sequence in the male fertility gene and identifying a cell comprising an alteration in the target sequence. Also disclosed are plants, plant cells, plant parts, and seeds comprising a male fertility gene with an alteration in a male fertility gene. Nucleic acid molecules comprising male fertility genes with at least one targeted modification therein, optimized nucleic acid molecules encoding endonucleases that are engineered double-strand-break-inducing agents and expression cassettes, host cells, and plants comprising one or more of the nucleic acid molecules are further disclosed.

1 - 46 . (canceled) 
     
     
         47 . A plant produced by a method for making a targeted modification in a male fertility gene in the genome of a plant, said method comprising: (a) contacting at least one plant cell comprising, in a MS26 male fertility gene, a target sequence comprising SEQ ID NO.: 1 with an engineered meganuclease that is capable of inducing a double-strand break at the target sequence in the MS26 male fertility gene, wherein the engineered meganuclease is modified to specifically cut at the target sequence, and wherein the engineered meganuclease no longer cuts at its wild-type meganuclease target sequence; and (b) identifying at least one cell from step (a) comprising an alteration in its genome at the target sequence wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) through (iii); wherein the alteration of the male fertility gene is a null mutation. 
     
     
         48 . A descendant of the plant of  claim 47 , wherein the descendant comprises the alteration. 
     
     
         49 . The plant of  claims 47  or  48 , wherein the targeted modification is a null mutation in the MS26 male fertility gene. 
     
     
         50 . The plant of  claims 47  or  48 , wherein the plant is homozygous for the alteration. 
     
     
         51 . The plant of  claims 47  or  48 , wherein the plant is male sterile. 
     
     
         52 . The plant of  claims 47  or  48 , wherein the plant is selected from the group consisting of maize,  sorghum , rice, wheat, rye, barley, millet and oat. 
     
     
         53 . A plant comprising a targeted modification in a male fertility gene in its genome, wherein the targeted modification is a targeted mutation or the insertion of a transgene, and wherein the male fertility gene is a MS26 male fertility gene. 
     
     
         54 . The plant of  claim 53 , wherein the targeted modification is a null mutation in the MS26 male fertility gene. 
     
     
         55 . The plant of  claim 53 , wherein the plant is homozygous for the alteration. 
     
     
         56 . The plant of  claim 53 , wherein the plant is male sterile. 
     
     
         57 . The plant of  claim 53 , wherein the plant is selected from the group consisting of maize,  sorghum , rice, wheat, rye, barley, millet and oat. 
     
     
         58 . A plant comprising an expression construct, said expression construct comprising a promoter operably linked to a nucleotide sequence encoding an engineered meganuclease that is capable of inducing a double-strand break in a MS26 male fertility gene comprising a target sequence comprising SEQ ID NO.: 1, wherein the engineered meganuclease is modified to specifically cut at said target sequence, and wherein the engineered meganuclease no longer cuts at its wild-type meganuclease target sequence, and wherein said promoter is capable of driving expression of an operably linked nucleotide sequence in a plant cell. 
     
     
         59 . The plant of  claim 58 , wherein the nucleotide sequence encoding an engineered meganuclease comprises a coding sequence encoding a DNA binding domain of selected from the group consisting of:
 (a) nucleotides 100-261 and nucleotides 661-822 of SEQ ID NO: 4;   (b) nucleotides 70-231 and nucleotides 631-792 of SEQ ID NO: 5;   (c) nucleotides 70-231 and nucleotides 820-981 of SEQ ID NO: 6, or 7; and,   (d) a degenerate coding sequence of (a), (b), or (c).   
     
     
         60 . The plant of  claim 59 , wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOS: 4, 5, 6, and 7. 
     
     
         61 . The plant of  claim 47  or  53 , wherein plant comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42. 
     
     
         62 . The plant of  claim 47  or  53 , wherein plant comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56 and 57. 
     
     
         63 . The plant of  claim 47  or  53 , wherein the plant comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 and 78. 
     
     
         64 . A progeny plant or seed derived from the plant of  claim 61 ,  62  or  63 , wherein the progeny plant comprising a targeted modification in a MS26 male fertility gene.