Granted Patent B2

US 12,543,674 · App. 17/594,386 · Granted Feb 10, 2026

Inventors: Jon Aaron Tucker Reinders (Clive, IA); Huaxun Ye (Johnston, IA)

Assignee: PIONEER HI-BRED INTERNATIONAL, INC.

A01H4/008A01H1/022A01H1/026A01H5/08C12N15/823C12N15/8233C12N15/8261

Abstract

Claims (20)

1 . A method of obtaining genome-modified, doubled haploid plant progeny, the method comprising:

(a) obtaining a first plant transformed with one or more expression cassettes comprising:

(i) a polynucleotide sequence encoding a morphogenic developmental polypeptide, wherein the morphogenic developmental polypeptide is a WUS/WOX homeobox polypeptide, a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a LEC1 polypeptide;

(ii) a polynucleotide sequence encoding an embryogenesis factor, wherein the embryogenesis factor is a maize cyclin delta-2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 21; and

(iii) a polynucleotide sequence encoding a Cas polypeptide, wherein the Cas polypeptide enables genomic modification of a DNA target site in the first plant, wherein at least one guide RNA is provided for the Cas polypeptide, the guide RNA comprising a region complementary to the DNA target site;

wherein the Cas polypeptide and the guide RNA form a complex that binds and modifies the DNA target site resulting in a modified target site;

(b) obtaining a second plant;

(c) pollinating the first plant with pollen from the second plant;

(d) obtaining embryogenic haploid progeny, wherein the haploid progeny comprises the genome of the first plant but not the second plant, and wherein the maize cyclin delta-2 polypeptide stimulates an embryogenic cell fate resulting in the embryogenic haploid progeny;

(e) obtaining doubled haploid progeny from the haploid progeny, wherein the maize cyclin delta-2 polypeptide stimulates diploidization of the haploid progeny, resulting in the doubled haploid progeny; and

(f) selecting genome-modified, doubled haploid plant progeny,

wherein the maize cyclin delta-2 polypeptide increases the frequency of embryogenic cell fate and diploidization in the haploid progeny relative to a non-treated control.

2 . The method of claim 1 , wherein the Cas polypeptide is a Cas9 nuclease, a Cas alpha nuclease, a Cpf1 nuclease, a dCas9-FokI, a dCpf1-FokI, a chimeric Cas9-cytidine deaminase, a chimeric Cas9 adenine deaminase, a nickase Cas9 (nCas9), a chimeric dCas9 non-FokI nuclease, or a dCpf1-non-FokI nuclease.

3 . The method of claim 1 , wherein the second plant comprises a marker gene selected from the group comprising of GUS, PMI, PAT, GFP, RFP, CFP, C1, CRC, R-nj, R1-scm, and anthocyanin pigments.

4 . The method of claim 1 , further comprising regenerating a plant from the doubled haploid progeny.

5 . The method of claim 1 , wherein the polynucleotide sequence encoding the maize cyclin delta-2 polypeptide is stably incorporated into the genome of the first plant.

6 . The method of claim 1 , wherein the maize cyclin delta-2 polypeptide of the first plant stimulates reprogramming in egg cells promoting the embryogenic cell fate in the egg cells.

7 . The method of claim 6 , wherein the maize cyclin delta-2 polypeptide of the first plant stimulates maternal haploid induction and renders egg cells to become parthenogenic egg cells having only the maternal genome.

8 . The method of claim 1 , wherein the polynucleotide sequence encoding the maize cyclin delta-2 polypeptide is operably linked to an egg cell promoter.

9 . The method of claim 1 , wherein the one or more expression cassettes further comprise an excision component for excision of the one or more polynucleotide sequences encoding the one or more morphogenic developmental polypeptides and/or the polynucleotide sequence encoding the Cas polypeptide.

Assignments (1)

Continuity (3)

References Cited (139)

Cited By (1)

US 12,735,718