Methods and compositions for rapid plant transformation
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.
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;
(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, wherein the expression occurs in the absence of exogenous cytokinin or in the presence of exogenous cytokinin at a final concentration of 0.1 mg/L or less; 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;
(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, wherein the expression occurs in the absence of exogenous cytokinin or in the presence of exogenous cytokinin at a final concentration of 0.1 mg/L or less; and
(c) germinating the somatic embryo to form the transgenic Poaceae plant;
wherein the WUS/WOX homeobox polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and
wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 18, 20, 63, 65, or 67; or the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to 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;
(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, wherein the expression occurs in the absence of exogenous cytokinin or in the presence of exogenous cytokinin at a final concentration of 0.1 mg/L or less; 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 comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant.
19 . The method of claim 17 , 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.
20 . The method of claim 19 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter.
21 . The method of claim 17 , wherein (c) is performed in the presence of exogenous cytokinin.
22 . The method of claim 17 , 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.
23 . The method of claim 20 , 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.
24 . The method of claim 22 , 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.
25 . The method of claim 1 , wherein the WUS/WOX homeobox polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and
wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 18, 20, 63, 65, or 67; or the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.
26 . The method of claim 17 , wherein the WUS/WOX homeobox polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15;
and wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises an amino acid sequence comprising at least 95% sequence identity to any of SEQ ID NO: 18, 20, 63, 65, or 67; or the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by a nucleotide sequence comprising at least 95% sequence identity to any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.
27 . The method of claim 4 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised.
28 . The method of claim 12 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised.
29 . The method of claim 20 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised.
30 . The method of claim 9 , wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and
wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 18, 20, 63, 65, or 67; or the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.
31 . The method of claim 25 , wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and
wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 18, 20, 63, 65, or 67, or the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by a nucleotide sequence comprising the nucleotide set forth in any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.
32 . The method of claim 26 , wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or the WUS/WOX homeobox polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and
wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises the amino acid sequence set forth in any of SEQ ID NO: 18, 20, 63, 65, or 67; or the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68.