Methods and compositions for isolating, identifying and characterizing monocot plastidic accase herbicide tolerant mutations using a model system

A method of producing an acetyl-CoA carboxylase (ACCase) enzyme that is tolerant to at least one herbicide, comprising:

• a. providing a yeast knockout complement comprising an ACCase-deficient yeast into which has been introduced a nucleic acid encoding a chimeric ACCase, said chimeric ACCase comprising an N-terminal region and a C-terminal region, wherein the C-terminal region comprises a wild-type herbicide sensitive sensitivity region (HSR) of a monocot plastidic ACCase of an Oryza species, and wherein the chimeric ACCase being herbicide sensitive;
• b. mutagenizing said yeast knockout complement under conditions that permit mutagenesis of at least one codon of the nucleic acid encoding the HSR of the chimeric ACCase to form a mutagenized yeast, and growing the mutagenized yeast thereby forming a library of mutagenized yeast colonies;
• c. culturing the mutagenized yeast colonies in the presence of at least one ACCase-inhibiting herbicide to form treated colonies;
• and
• d. isolating from at least one of said treated colonies at least one mutagenized yeast that grows in the presence of the herbicide, wherein the mutagenized yeast that grows in the presence of the herbicide comprises a mutagenized chimeric ACCase that has a tolerance to the herbicide that is greater than that exhibited by the chimeric ACCase of step (a),
• wherein the method is a high throughput method in which one trained worker can generate and screen at least 500 different herbicide-sensitive-region (HSR)-variant ACCases, each from its own cell colony, in one month.