Methods for enhancing genome engineering efficiency

A maize plant cell comprising

• a) a polynucleotide encoding a RKD2 polypeptide and a polynucleotide encoding a RKD4 polypeptide;
• and
• b) a transgene of interest and/or a genome engineering component;
• wherein the polynucleotide encoding the RKD2 polypeptide or the RKD4 polypeptide is operably linked to a heterologous promoter, which is either directly chemically inducible or indirectly chemically inducible, and
• wherein the nucleic acid encoding the RKD2 polypeptide and the RKD4 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 3 or 4, a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 3 or 4 over a full length of the nucleic acid, a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 1, and a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 1 over a full length of the nucleic acid.

A maize plant cell comprising a DNA construct, wherein the DNA construct comprises

• a) a nucleic acid comprising a polynucleotide encoding a RKD2 and a RKD4 polypeptide operably linked to a chemically inducible bidirectional promoter,
• b) a reporter gene operably linked to the bidirectional promoter, and
• c) a third recombinant gene encoding a transcription factor operably linked to a strong constitutive promoter,
• wherein the nucleic acid encoding the RKD2 polypeptide and the RKD4 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 3 or 4, a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 3 or 4 over a full length of the nucleic acid, a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 1, and a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 1 over a full length of the nucleic acid.

A method of using a nucleic acid comprising a polynucleotide encoding a RKD2 polypeptide and a RKD4 polypeptide, a recombinant gene comprising the nucleic acid, a DNA construct comprising the nucleic acid for improving the efficiency of maize plant regeneration or increasing the regeneration ability of a maize plant cell upon chemical induction, the method comprising:

• (a) introducing into the maize plant cell
• (i) a nucleic acid comprising a polynucleotide sequence encoding the RKD2 polypeptide and the RKD4 polypeptide, a recombinant gene comprising the nucleic acid, or a DNA construct comprising the nucleic acid;
• and
• (ii) a transgene of interest and/or a genome engineering component;
• (b) optionally, cultivating the maize plant cell under conditions allowing the synthesis of the RKD2 polypeptide or the RKD4 polypeptide from the nucleic acid after chemical induction;
• and
• (c) optionally, cultivating the maize plant cell under conditions allowing the genetic modification of the genome of said maize plant cell by integration of the transgene of interest and activity of the genome engineering component in the presence of the RKD2 polypeptide and the RKD4 polypeptide;
• wherein the polynucleotide sequence encoding the RKD2 polypeptide and the RKD4 polypeptide is operably linked to a heterologous promoter, which is either directly chemically inducible or indirectly chemically inducible,
• wherein the nucleic acid encoding the RKD2 polypeptide and the RKD4 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 3 or 4, a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 3 or 4 over a full length of the nucleic acid, a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
• 1, and a nucleic acid comprising a nucleotide sequence at least 95% identity to SEQ ID NO:
• 1 over a full length of the nucleic acid.