Haploid maize transformation
Inventors
Petolino, Joseph F. • Strange, Tonya L. • Samuel, Jayakumar Pon • Blue, Ryan C. • Simpson, Matthew A.
Assignees
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Publication Number
US-12644127-B2
Publication Date
2026-06-02
Expiration Date
Abstract
Disclosed are methods for transformation of an androgenic-derived, haploid cell line with a site-specific nuclease. In some embodiments, the androgenic-derived, haploid cell line is a maize microspore-derived plant tissue culture. In addition, the disclosure provides a method for modifying, e.g., by mutating or targeting and integrating donor DNA into, a specific locus of a haploid or dihaploid tissue genome. The disclosure further provides methods for regenerating a whole plant from the haploid or dihaploid tissue that contains either the mutation at a specific genomic locus or a donor DNA integrated within a specific genomic locus may be obtained from the subject disclosure.
Core Innovation
The invention provides a method for modifying a maize genome by zinc finger nuclease site-specific targeted mutagenesis in microspore-derived, transformation-competent haploid callus tissue. The method starts by crossing an elite maize line with a different maize line having high microspore culture response to produce a hybrid maize line having elite performance characteristics and high microspore culture response. Microspore-derived, transformation-competent callus tissue is produced from progeny of the hybrid maize line, and the callus is assayed and determined to be haploid, comprising a paternal haploid tissue genome from the progeny.
A polynucleotide encoding a site-specific nuclease is delivered to the haploid callus, resulting in double strand cleavage of the haploid genome of the callus by particle bombardment. The site-specific nuclease targeting specificity is engineered to target the modified genomic sequence with at least an 8-fold increased level of targeted mutagenesis as compared to non-transformed callus. After delivery, the haploid callus genome is confirmed to be modified by the encoded site-specific nuclease.
The disclosure further includes confirming modification using PCR, Southern blot, Northern blot, Western blot, ELISA, and Next Generation Sequencing. Additional disclosed refinements address donor polynucleotide delivery and stable integration into a target region, and chromosome doubling with a chromosome doubling agent to regenerate dihaploid maize tissue into a homozygous dihaploid maize plant. The resulting dihaploid maize plant is then used in breeding by producing F1 progeny through crossing with a different maize line and optionally performing backcrossing with additional selection.
Claims Coverage
The disclosed claim set centers on one independent claim describing a full workflow for zinc finger nuclease site-specific targeted mutagenesis in maize haploid callus. Dependent claims further refine confirming assays, donor polynucleotide constraints and integration, chromosome doubling and regeneration, and downstream crossing/backcrossing selection. The inventive core is characterized by engineered nuclease targeting specificity with an at least 8-fold increased targeted mutagenesis level in modified genomic sequence versus non-transformed callus.
Crossing elite lines to generate hybrid maize with high microspore culture response
Crossing an elite maize line with a different maize line having high microspore culture response to produce a hybrid maize line having elite performance characteristics and high microspore culture response.
Producing transformation-competent microspore-derived haploid callus
Producing microspore-derived, transformation-competent callus tissue from progeny of the hybrid maize line; isolating a maize microspore-derived, transformation-competent haploid callus comprising a paternal haploid tissue genome from the progeny; assaying and determining that the callus is haploid.
Particle bombardment delivery of zinc finger nuclease polynucleotide to haploid genome
Delivering a polynucleotide encoding a site-specific nuclease to the transformation-competent haploid callus resulting in double strand cleavage of the haploid genome of the callus by particle bombardment.
Engineered targeting specificity for increased targeted mutagenesis and confirmation of modification
Confirming that the haploid callus genome is modified by the encoded site-specific nuclease, wherein the site-specific nuclease's targeting specificity has been engineered to target the modified genomic sequence with at least an 8-fold increased level of targeted mutagenesis as compared to non-transformed callus.
Confirming genome modification using PCR, blots, protein expression assays, or NGS
Confirming that the haploid callus genome is modified using one or more of PCR, Southern blot, Northern blot, Western blot, ELISA, or Next Generation Sequencing assays.
Donor polynucleotide sequence identity constraint for target region
The donor polynucleotide comprises one or two domains, where each domain is at least 85% identical to a sequence in the genomic DNA target region of the haploid callus genome.
Stably integrating donor polynucleotide into target region
Stably integrating the donor polynucleotide into a target region of the haploid tissue genome and confirming integration into the target region.
Chromosome doubling to regenerate homozygous dihaploid plants
Treating haploid callus with a chromosome doubling agent to produce dihaploid maize tissue containing a stably integrated donor polynucleotide, and regenerating it into a homozygous dihaploid maize plant.
Breeding modified plants via F1 crossing and optional backcrossing selection
Crossing the dihaploid maize plant with a different parent maize line to produce F1 progeny, selecting F1 plants carrying the genome modification, and optionally performing backcrossing with repeated selection to obtain additional modified progeny.
Overall, the independent claim covers production of haploid microspore-derived transformation-competent callus from elite-line crosses, particle bombardment delivery of a zinc finger nuclease polynucleotide, and confirmation of modification with engineered targeting specificity that provides at least an 8-fold increased targeted mutagenesis level relative to non-transformed callus. Dependent claims extend coverage to specific confirmation assays, donor polynucleotide sequence identity constraints and stable integration, chromosome doubling and regeneration into homozygous dihaploid plants, and downstream F1 crossing with optional backcrossing for selecting additional modified progeny.
Stated Advantages
Increased targeted mutagenesis level, wherein engineered targeting specificity targets the modified genomic sequence with at least an 8-fold increased level compared to non-transformed callus.
Documented Applications
Applying the method to modify a maize genome using zinc finger nuclease site-specific targeted mutagenesis in microspore-derived haploid callus.
Generating dihaploid maize plants with a stably integrated donor polynucleotide followed by breeding to produce F1 progeny and optionally backcross progeny plants carrying the genome modification.
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