Methods and compositions for producing male sterile plants
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. 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.
2. The method of claim 1 , wherein a plant that is homozygous for the null mutation is male sterile.
3. The method of claim 1 , further comprising selfing the plant and selecting a progeny plant resulting therefrom, wherein said progeny plant is homozygous for the alteration.
4. The method of claim 1 , further comprising crossing the plant with a second fertile plant comprising a null mutation in the male fertility gene and selecting a progeny plant resulting therefrom, wherein said progeny is male sterile.
5. The method of claim 1 , wherein the alteration comprises insertion of a transgene comprising a polynucleotide of interest.
6. The method of claim 5 , wherein the transgene further comprises a promoter operably linked to the polynucleotide of interest, and wherein the promoter is capable of driving the expression of the polynucleotide of interest in a plant.
7. The method of claim 1 , wherein the plant is selected from the group consisting of maize, sorghum, rice, wheat, rye, barley, millet and oat.
8. The method of claim 1 , wherein the engineered meganuclease is derived from I-CreI.
9. The method of claim 1 , wherein step (a) further comprises introducing into the at least one plant cell a nucleic acid construct comprising a nucleotide sequence encoding the engineered meganuclease.
10. The method of claim 9 , wherein the nucleotide sequence is the nucleotide sequence set forth in SEQ ID NO: 4, 5, 6, or 7.
11. The method of claim 10 , wherein the nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the engineered meganuclease, wherein the promoter is capable of driving expression of the nucleotide sequence in a plant cell.
12. The method of claim 11 , wherein the promoter is a maize ubiquitin promoter.
13. The method of claim 10 , wherein the nucleic acid construct further comprises an operably linked nucleotide sequence encoding a nuclear localization signal.
14. The method of claim 13 , wherein the nuclear localization signal comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, and 3.