초록 ▼

The present invention relates to the production of a non-transgenic plant resistant or tolerant to a herbicide of the phosphonomethylglycine family, e.g., glyphosate. The present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or ep

The present invention relates to the production of a non-transgenic plant resistant or tolerant to a herbicide of the phosphonomethylglycine family, e.g., glyphosate. The present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS). The mutated protein, which substantially maintains the catalytic activity of the wild-type protein, allows for increased resistance or tolerance of the plant to a herbicide of the phosphonomethylglycine family, and allows for the substantially normal growth or development of the plant, its organs, tissues or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide.

대표청구항 ▼

1. A method for producing a non-transgenic, herbicide resistant or tolerant plant comprising:(a) introducing into plant cells a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene to produce plant cells with a mutant EPSPS gene that expresses a EPSPS protein that is mutated at

1. A method for producing a non-transgenic, herbicide resistant or tolerant plant comprising:(a) introducing into plant cells a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene to produce plant cells with a mutant EPSPS gene that expresses a EPSPS protein that is mutated at one or more amino acid positions, said positions selected from the group consisting of Leu173, Ala179, Met180, Arg181, Ser98, Ser255 and Leu198 in the Arabidopsis EPSPS protein or at an analogous amino acid residue in an EPSPS paralog; (b) identifying a plant cell having substantially normal growth as compared to a corresponding wild-type plant cell in the presence of glyphosate; and (c) regenerating a non-transgenic herbicide resistant or tolerant plant having a mutated EPSPS gene from said plant cell. 2. A method for producing a non-transgenic, herbicide resistant or tolerant plant comprising:(a) introducing into plant cells a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene to produce plant cells with a mutant EPSPS gene that expresses a mutant EPSPS protein that is mutated at one or more amino acid positions, said positions selected from the group consisting of Leu173, Ala179, Met180, Arg181, Ser98, Ser255 and Leu198 in the Arabidopsis EPSPS protein or at an analogous amino acid residue in an EPSPS paralog; (b) identifying a plant cell having mutant EPSPS protein that has substantially the same catalytic activity as compared to a corresponding wild type EPSPS protein in the presence of glyphosate; and (c) regenerating a non-transgenic herbicide resistant or tolerant plant having a mutated EPSPS gene from said plant cell. 3. The method according to claim 1 or 2 in which the recombinagenic oligonucleobase is a mixed duplex nucleotide or a SSMOV.4. The method according to claim 3 in which the mixed duplex nucleotide contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of the first fragment of the target EPSPS gene and a second homologous region which has a sequence identical to the sequence of at least 6 based pairs of a second fragment of the target EPSPS gene, and an intervening region which contains at least one nucleobase heterologous to the target EPSPS gene, which intervening region connects the first and second homologous region.5. The method according to claim 1 or 2 in which the recombinagenic oligonucleobase is introduced by electroporation.6. The method according to claim 1 in which the amino acid positions are selected from the group consisting of Leu97, Ala103, Met104, Arg105, Ser23, Ser179 and Leu122 in the Zea mays paralog.7. The method according to claim 1 in which the amino acid positions are selected from the group consisting of Leu169, Ala175, Met178, Arg177, Ser94, Ser251 and Leu194 in a Brassica sp paralog.8. The method according to claim 1 in which the amino acid positions are selected from the group consisting of Leu169, Ala175, Met176, Arg177, Ser94, Ser251 and Leu194 in the Petunia hybrida paralog.9. The plant according to claim 1 or 2 in which the plant cells are selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus, apple, lettuce, peas, lentils, grape, turf grasses and Brassica sp.10. The method according to claim 2 in which the amino acid positions are selected from the group consisting of Leu97, Ala103, Met104, Arg105, Ser23, Ser179 and Leu122 in the Zea mays paralog.11. The method according to claim 2 in which the amino acid positions are selected from the group consisting of Leu169, Ala175, Met176, Arg177, Ser94, Ser251 and Leu194 in a Brassica sp paralog.12. The method according to claim 2 in which the amino acid positions are selected from the group consisting of Leu189, Ala175, Met176, Arg177, Ser94, Ser251 and Leu194 in the Petunia hybrida paralog.13. A method for producing a non-transgenic, herbicide resistant or tolerant plant comprising:(a) introducing into plant cells a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene to produce plant cells with a mutant EPSPS gene that expresses an EPSPS protein that is mutated in two amino acid positions, said positions selected from the group consisting of Thr178 and Pro182, in the Arabidopsis EPSPS protein or at an analogous amino acid residue in an EPSPS paralog wherein the Thr178 is changed to Val or Leu and Pro182 is changed to Ser; (b) identifying a plant cell having substantially normal growth as compared to a corresponding wild-type plant cell in the presence of glyphosate; and (c) regenerating a non-transgenic herbicide resistant or tolerant plant having a mutated EPSPS gene from said plant cell. 14. The method according to claim 13 in which the amino acid positions are Thr102 and Pro106 in the Zea mays paralog.15. The method according to claim 13 in which the amino acid positions are Thr174 and Pro178 in a Brassica sp paralog.16. The method according to claim 13 in which the amino acid positions are Thr174 and Pro178 in the Petunia hybrida paralog.