CPC C12N 15/102 (2013.01) [C12N 5/0025 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2500/02 (2013.01); C12N 2500/14 (2013.01); C12N 2500/16 (2013.01); C12N 2800/80 (2013.01)] | 4 Claims |
1. A method for making a maize plant protoplast having a genomic modification comprising:
(a) providing genome editing molecules to a maize plant protoplast lacking a cell wall wherein the genome editing molecules are designed for homology-directed repair of a target gene in the plant cell's genome and comprise: (i) an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof,
(b) exposing the maize plant protoplast from step (a) to plant cell culture medium comprising a reactive oxygen species (ROS) concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar and Ca2+ and/or Mg2+ at a concentration of about 40 mM to 150 mM for at least one hour to effect homology-directed repair of the target gene in the plant cell's genome at a frequency that is increased by at least 2-fold in comparison to a control method wherein a control plant cell is provided with the genome editing molecules but is not subjected to a ROS concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar, wherein the ROS concentration lowering agent is a ROS scavenging agent or an antioxidant; and,
(c) isolating, selecting, identifying, and/or propagating a maize plant protoplast comprising the genome modification, thereby making the maize plant protoplast having a genomic modification.
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