Use of mixed duplex oligonucleotides to effect localized genetic changes in plants

A method of making a localized, non-selectable mutation in a target gene in a plant cell comprising the steps of:

• a. introducing into the cells of a population of cells a mixture of a first recombinagenic oligonucleobase and a second reombinagenic oligonucleobase wherein:
• i. the first recombinagenic oligonucleobase contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of a first target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the first target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening region connects the first homologous region and the second homologous region, and
• ii. the second recombinagenic oligonucleobase contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of a second target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the second target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening region connects the first homologous region and the second homologous region;
• b. selecting cells from the population having a selectable mutation located between the first and the second fragments of the first target gene from the population;
• and
• c. identifying a selected cell having a non-selectable mutation located between the first fragment and the second fragment of the second target cell.

A method of making a localized mutation in a target gene in a plant cell comprising the steps of:

• a. digesting a plant part with cellulase such that plant cell protoplasts are formed;
• b. suspending the protoplasts in a solution comprising a recombinagenic oligonucleobase which contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of the target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening region connects the first homologous region and the second homologous region;
• c. electroporating the suspension such that the recombinagenic oligonucleobase enters a protoplast of the suspension;
• d. culturing the protoplast;
• and
• e. identifying a progeny of the protoplast having a mutation located between the first and second fragments of the target gene.