Methods and compositions for rapid plant transformation

Patent metadata

Field	Value
Publication number	US-11330776-B2
Application number	US-201615249318-A
Country	US
Kind code	B2
Filing date	Aug 26, 2016
Priority date	Oct 30, 2015
Publication date	May 17, 2022
Grant date	May 17, 2022

Abstract

The disclosure pertains to methods and compositions for the rapid and efficient transformation of plants. The disclosure further provides methods for producing a transgenic plant, comprising (a) transforming a cell of an explant with an expression construct comprising (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding a polypeptide comprising two AP2-DNA binding domains; or (iii) a combination of (i) and (ii); and (b) allowing expression of the polypeptide of (a) in each transformed cell to form a regenerable plant structure in the absence of exogenous cytokinin, wherein no callus is formed; and (c) germinating the regenerable plant structure to form the transgenic plant. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

First claim

That which is claimed:

1. A method for producing a transgenic Poaceae plant, comprising:
(a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
(i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and
(ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and

(b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and
(c) germinating the somatic embryo to form the transgenic Poaceae plant.

2. The method of claim 1 , wherein germinating comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant.

3. The method of claim 1 , wherein the expression construct further comprises a nucleotide sequence encoding a site-specific recombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153.

4. The method of claim 3 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter.

5. The method of claim 1 , wherein (c) is performed in the presence of exogenous cytokinin.

6. The method of claim 1 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is operably linked to a promoter selected from an inducible promoter, a developmentally regulated promoter, or a constitutive promoter.

7. The method of claim 4 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and
the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.

8. The method of claim 6 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and
the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.

9. A method for producing a transgenic Poaceae plant, comprising:
(a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
(i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and
(ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and

(b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and
(c) germinating the somatic embryo to form the transgenic Poaceae plant;

wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or wherein the WUS/WOX homeobox polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15;
wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide comprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65, or 67; or wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.

10. The method of claim 9 , wherein germinating comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant.

11. The method of claim 9 , wherein the expression construct further comprises a nucleotide sequence encoding a site-specific recombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153.

12. The method of claim 11 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter.

13. The method of claim 9 , wherein (c) is performed in the presence of exogenous cytokinin.

14. The method of claim 9 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding a polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is operably linked to an inducible promoter, a developmentally regulated promoter, or a constitutive promoter.

15. The method of claim 12 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and
the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34.

16. The method of claim 14 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and
the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.

17. A method for producing a transgenic Poaceae plant comprising:
(a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
(i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and
(ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and

(b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and
(c) germinating the somatic embryo of (b) for about 14 to about 60 days to form a plantlet; and
(d) allowing the plantlet of (c) to grow into a Poaceae plant.

18. The method of claim 17 , wherein germinating co

Assignees

• Pioneer Hi Bred Int

Inventors

• Anand Ajith
• Arling Maren L
• Da Silva Conceicao Alexandre
• Gordon-Kamm William James
• Hastings Craig E
• Hoerster George J
• Jones Todd J
• Klein Theodore Mitchell
• La Rota Carlos M
• Lowe Keith S
• Tiwari Shiv Bahadur
• Wang Ning
• Wu Xinli Emily

Classifications

• C07K14/415 Primary

  from plants · CPC title

• A01H4/008 Primary

  Methods for regeneration to complete plants · CPC title

• C12N15/8216

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title

• C12N15/821

  Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers · CPC title

• C12N15/8201

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title