Methods for clonal plant production
The methods disclosed herein provide for clonal plant production using a morphogenic gene to produce non-transgenic and transgenic plants wherein the morphogenic gene does not integrate into the genome of the plant.
1. A method of producing a transgenic plant comprising:
providing to a plurality of haploid embryos or haploid embryo-like structures a trait gene expression cassette and a morphogenic gene expression cassette, the plurality of haploid embryos or haploid embryo-like structures comprising one or more first haploid embryos or embryo-like structures and one or more second haploid embryos or embryo-like structures, wherein at least one first haploid embryo or embryo-like structure includes the morphogenic gene expression cassette and expresses a morphogenic polypeptide;
inducing somatic embryogenesis in the one or more second haploid embryos or embryo-like structures resulting in one or more somatic embryos, wherein somatic embryogenesis is induced by contacting the one or more second haploid embryos or embryo-like structures with the morphogenic polypeptide expressed by the at least one first haploid embryo or embryo-like structure and translocated to the one or more second haploid embryos or embryo-like structures, wherein the one or more second haploid embryos or embryo-like structures do not include the morphogenic gene expression cassette;
selecting a somatic embryo comprising the trait gene expression cassette and lacking the morphogenic gene expression cassette;
contacting the selected somatic embryo with a chromosome doubling agent for a period sufficient to generate a doubled haploid embryo; and
regenerating a transgenic plant from the selected doubled haploid embryo, the transgenic plant comprising the trait gene expression cassette and lacking the morphogenic gene expression cassette.
2. The method of claim 1 , wherein the trait gene expression cassette and the morphogenic gene expression cassette are provided via particle gun delivery.
3. The method of claim 2 , wherein the trait gene expression cassette and the morphogenic gene expression cassette are delivered to the plurality of haploid embryos or haploid embryo-like structures simultaneously.
4. The method of claim 2 , wherein the trait gene expression cassette and the morphogenic gene expression cassette are delivered to the plurality of haploid embryos or haploid embryo-like structures sequentially.
5. The method of claim 1 , wherein the trait gene expression cassette and the morphogenic gene expression cassette are provided via bacterial-mediated delivery.
6. The method of claim 5 , wherein the bacterial-mediated delivery comprises simultaneously contacting the plurality of haploid embryos or the haploid embryo-like structures with a T-DNA containing the trait gene expression cassette in a first bacterial strain and a T-DNA containing the morphogenic gene expression cassette in a second bacterial strain.
7. The method of claim 5 , wherein the bacterial-mediated delivery comprises contacting the plurality of haploid embryos or the haploid embryo-like structures with a T-DNA containing the trait gene expression cassette and a T-DNA containing the morphogenic gene expression cassette in one bacterial strain.
8. The method of claim 1 , wherein the morphogenic gene expression cassette comprises:
(i) a nucleotide sequence encoding a WUS/WOX polypeptide; or
(ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; or
(iii) a combination of (i) and (ii).
9. The method of claim 8 , wherein the nucleotide sequence encoding the WUS/WOX polypeptide is selected from the group consisting of WUS1, WUS2, WUS3, WOX2 A, WOX4, WOX5, and WOX9.
10. The method of claim 8 , wherein the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from the group consisting of BBM2, BMN2, and BMN3 or the Ovule Development Protein 2 (ODP2) polypeptide is ODP2.
11. The method of claim 1 , wherein the trait gene expression cassette comprises:
a trait gene selected from the group consisting of a gene conferring pest resistance, a gene conferring herbicide resistance, a gene conferring stress tolerance, a gene conferring drought resistance, a gene conferring nitrogen use efficiency, a gene conferring disease resistance, and a gene conferring an ability to alter a metabolic pathway.
12. The method of claim 11 , wherein the trait gene expression cassette further comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide.
13. The method of claim 12 , wherein the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from the group consisting of BBM2, BMN2, and BMN3 or the Ovule Development Protein 2 (ODP2) polypeptide is ODP2.
14. The method of claim 1 , wherein the trait gene expression cassette or the morphogenic gene expression cassette further comprises a polynucleotide encoding a site-specific nuclease.
15. The method of claim 14 , wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Cas nuclease.
16. The method of claim 15 , wherein the CRISPR-Cas nuclease is a Cas9 or a Cpf1 nuclease.
17. The method of claim 14 , wherein the site-specific nuclease effects an insertion, a deletion, or a substitution mutation.
18. The method of claim 15 , further comprising providing a guide RNA expressed from the trait gene expression cassette or the morphogenic gene expression cassette.
19. The method of claim 18 , wherein the guide RNA and CRISPR-Cas nuclease form a ribonucleoprotein complex.
20. The method of claim 6 , wherein the first bacterial strain and the second bacterial strain are present in a 50:50 ratio, a 75:25 ratio, 90:10 ratio, a 95:5 ratio, or a 99:1 ratio.
21. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises a promoter selected from a group consisting of ZM-PLTP, SB-PLTP1, ZM-FBP1, ZM-RFP, ZM-APMP, ZM-RfeSP, ZM-CRR6, ZM-GLYK, ZM-CAB7, ZM-UBR, ZM-HBP, ZM-PSAN, ZM-SDR, AXIG1, DR5, ZM-PLTP1, ZM-PLTP2, SB-PLTP2, SB-PLTP3, SI-PLTP1, OS-PLTP1, OS-PLTP2, ZM-LGL PRO, ZM-LEA14 PRO, ZM-LEA34-D PRO, ZM-SDR PRO (long), OS-SDR PRO, SB-SDR PRO, and GM-EF1 A PRO or from a group consisting of SEQ ID NO: 35-105, and 106.
22. The method of claim 21 , wherein the morphogenic gene expression cassette comprises a PLTP promoter operably linked to the nucleotide sequence encoding a functional WUS/WOX polypeptide.
23. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises an expression modulating element selected from the group consisting of SEQ ID NO: 107-173 and 174.
24. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises an enhancer selected from the group consisting of SEQ ID NO: 175-179 and 180.
25. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises an expression modulating element selected from the group consisting of SEQ ID NO: 107-173 and 174 and an enhancer selected from the group consisting of SEQ ID NO: 175-179 and 180.
26. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises three copies of an expression modulating element selected from the group consisting of SEQ ID NO: 107-173 and 174, wherein the three copies of the expression modulating element are the same or different expression modulating element.
27. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises three copies of an enhancer selected from the group consisting of SEQ ID NO: 175-179 and 180, wherein the three copies of the enhancer are the same or different enhancer.
28. The method of claim 8 , wherein the morphogenic gene expression cassette further comprises three copies of an expression modulating element selected from the group consisting of SEQ ID NO: 107-173 and 174 and the enhancer comprises three copies of an enhancer selected from the group consisting of SEQ ID NO: 175-179 and 180, wherein the three copies of the expression modulating element are the same or different expression modulating element and/or the three copies of the enhancer are the same or different enhancer.
29. The method of claim 1 , further comprising:
crossing the regenerated transgenic doubled haploid plant with a plant comprising a desired genotype/phenotype; and
growing offspring having the desired genotype/phenotype.
30. The method of claim 1 , wherein the plurality of haploid embryos are maternal haploid embryos.
31. The method of claim 30 , wherein the maternal haploid embryos are derived from pollination of a female parent plant with pollen from a haploid inducer male parent plant and the maternal haploid embryos result from paternal genome elimination.
32. The method of claim 31 , wherein the female parent plant is a hybrid plant.
33. The method of claim 1 , wherein the plurality of haploid embryos are microspore-derived haploid embryos.
34. The method of claim 1 , wherein the plurality of haploid embryo-like structures are microspore-derived haploid embryo-like structures.