Methods and compositions for targeted polynucleotide modification

A method for targeting the insertion of a polynucleotide of interest to a target site in a plant cell, wherein said target site comprises a first recombination site, said method comprising:

• a) introducing into said plant cell at least one heterologous polynucleotide encoding a cell proliferation factor and expressing said heterologous polynucleotide encoding said cell proliferation factor;
• b) introducing into said plant cell a transfer cassette comprising a second recombination site and said polynucleotide of interest, wherein the first and said second recombination sites are recombinogenic with respect to one another;
• and
• c) introducing into said plant cell a site-specific recombinase that recognizes and implements recombination at said first and said second recombination sites, thereby inserting said polynucleotide of interest at the target site.

A method for targeting the insertion of a polynucleotide of interest to a target site in a plant cell, wherein said target site comprises a first and a second recombination site, wherein said first and said second recombination sites flank a nucleotide sequence and are non-recombinogenic with respect to one another, said method comprising:

• a) introducing into said plant cell at least one heterologous polynucleotide encoding a cell proliferation factor and expressing said heterologous polynucleotide encoding said cell proliferation factor;
• b) introducing into said plant cell a transfer cassette comprising a third and a fourth recombination site flanking said polynucleotide of interest, wherein the third recombination site is recombinogenic with the first recombination site, and wherein the fourth recombination site is recombinogenic with the second recombination site;
• and
• c) introducing into said plant cell a site-specific recombinase that recognizes and implements recombination at the first, second, third, and fourth recombination sites;
• thereby replacing the nucleic acid sequence of the target site with the polynucleotide of interest from the transfer cassette.

A method to integrate multiple transfer cassettes at a target site in a plant cell, wherein said target site comprises at least a first and a second recombination site, said method comprising:

• a) introducing into said plant cell a first transfer cassette comprising in the following order:
• at least the first, a third, and the second recombination sites, wherein the first and the third recombination sites of the first transfer cassette flank a first polynucleotide of interest, and wherein said first, said second, and said third recombination sites are non-recombinogenic with respect to one another;
• b) introducing into said plant cell a first site-specific recombinase, wherein said site-specific recombinase recognizes and implements recombination at the first and the second recombination sites;
• c) introducing a second transfer cassette comprising at least the second and the third recombination sites, wherein the second and the third recombination sites of the second transfer cassette flank a second polynucleotide of interest;
• and
• d) introducing into said plant cell a second site-specific recombinase, wherein said second site-specific recombinase recognizes and implements recombination at the second and third recombination sites;
• whereby the first and the second transfer cassettes are integrated at the target site of the plant cell, and wherein said method further comprises introducing at least one heterologous polynucleotide encoding a cell proliferation factor into said plant cell and expressing said heterologous polynucleotide encoding said cell proliferation factor before or during the introduction of the first site-specific recombinase, the second site-specific recombinase, or both the first and the second site-specific recombinase.

A method to integrate multiple transfer cassettes at a target site in a plant cell, wherein said target site comprises in the following order at least a first, a second, and a third recombination site, wherein said first, said second, and said third recombination sites are non-recombinogenic with respect to one another, said method comprising:

• a) introducing into said plant cell a first transfer cassette comprising a first polynucleotide of interest flanked by the first and the second recombination sites;
• b) introducing into said plant cell a first site-specific recombinase, wherein said first site-specific recombinase recognizes and implements recombination at the first and the second recombination sites;
• c) introducing a second transfer cassette comprising a second polynucleotide of interest flanked by at least the second and the third recombination sites;
• and
• d) introducing into said plant cell a second site-specific recombinase, wherein said second site-specific recombinase recognizes and implements recombination at the second and third recombination sites;
• whereby the first and the second transfer cassettes are integrated at the target site of the plant cell, and wherein said method further comprises introducing at least one heterologous polynucleotide encoding a cell proliferation factor into said plant cell and expressing said heterologous polynucleotide encoding said cell proliferation factor before or during the introduction of the first site-specific recombinase, the second site-specific recombinase, or both the first and the second site-specific recombinase.