U.S. patent application number 10/136998 was filed with the patent office on 2003-12-25 for method of producing transgenic maize using direct transformation of commercially important genotypes.
Invention is credited to Bowman, Cindy, Cammack, Francis P., Carozzi, Nadine, Cassagne, Francis E., Clucas, Christopher P., Colbert, Terry Ray, Crossland, Lyle D., Dawson, John L., De Framond, Annick, Desai, Nalini, Dunder, Erik, Evola, Steve, Hornbrook, Albert R., Koziel, Michael, Kramer, Vance, Launis, Karen, Lewis, Kelly, Linder, James O., Meghji, Moez R., Merlin, Ellis, Miller, Robert L., Mousel, Alan W., Pace, Gary M., Pollini, Gilles, Rothstein, Steven J., Skillings, Bruce W., Suttie, Jan, Tanner, Andreas H., Warren, Gregory, Wright, Martha.
Application Number | 20030237117 10/136998 |
Document ID | / |
Family ID | 27533276 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030237117 |
Kind Code |
A1 |
Koziel, Michael ; et
al. |
December 25, 2003 |
Method of producing transgenic maize using direct transformation of
commercially important genotypes
Abstract
Methods for transformation of maize with nucleic acid sequences
of interest are disclosed. The method involves subjecting immature
zygotic embryos or Type I callus to high velocity microprojectile
bombardment. The method is capable of producing transformed maize
lines of commercial importance and their hybrid combinations.
Inventors: |
Koziel, Michael; (Raleigh,
NC) ; Desai, Nalini; (Chapel Hill, NC) ;
Lewis, Kelly; (Cary, NC) ; Kramer, Vance;
(Hillsborough, NC) ; Warren, Gregory; (Apex,
NC) ; Evola, Steve; (Cary, NC) ; Crossland,
Lyle D.; (Chesterfield, MO) ; Wright, Martha;
(Overland Park, KS) ; Merlin, Ellis; (Raleigh,
NC) ; Launis, Karen; (Franklinton, NC) ;
Rothstein, Steven J.; (Clive, IA) ; Bowman,
Cindy; (Raleigh, NC) ; Dawson, John L.;
(Greensboro, NC) ; Dunder, Erik; (Hillsborough,
NC) ; Pace, Gary M.; (Cary, NC) ; Suttie,
Jan; (Raleigh, NC) ; Carozzi, Nadine;
(Raleigh, NC) ; De Framond, Annick; (Research
Triangle Park, NC) ; Linder, James O.; (Owatonna,
MN) ; Miller, Robert L.; (Iowa City, IA) ;
Skillings, Bruce W.; (Ontario, CA) ; Mousel, Alan
W.; (Northfield, MN) ; Hornbrook, Albert R.;
(Normal, IL) ; Clucas, Christopher P.; (Rochelle,
IL) ; Meghji, Moez R.; (Bloomington, IL) ;
Tanner, Andreas H.; (Plaisance Du Touch, FR) ;
Cassagne, Francis E.; (Auch, FR) ; Pollini,
Gilles; (L'Isle En Dodon, FR) ; Colbert, Terry
Ray; (Fort Branch, IN) ; Cammack, Francis P.;
(Rochelle, IL) |
Correspondence
Address: |
WHITE & CASE LLP
PATENT DEPARTMENT
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
27533276 |
Appl. No.: |
10/136998 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10136998 |
Apr 30, 2002 |
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08438666 |
May 10, 1995 |
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6403865 |
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08438666 |
May 10, 1995 |
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08008374 |
Jan 25, 1993 |
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08008374 |
Jan 25, 1993 |
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07951715 |
Sep 25, 1992 |
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5625136 |
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07951715 |
Sep 25, 1992 |
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07772027 |
Oct 4, 1991 |
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07951715 |
Sep 25, 1992 |
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07659433 |
Feb 25, 1991 |
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07659433 |
Feb 25, 1991 |
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07573105 |
Aug 24, 1990 |
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Current U.S.
Class: |
800/320.1 ;
435/412; 435/468 |
Current CPC
Class: |
Y02A 40/146 20180101;
C07K 14/325 20130101; C12N 9/12 20130101; Y02A 40/162 20180101;
C12N 15/8286 20130101; C12N 9/88 20130101 |
Class at
Publication: |
800/320.1 ;
435/412; 435/468 |
International
Class: |
A01H 005/00; C12N
005/04; C12N 015/82 |
Claims
What is claimed is:
1. A method for producing stably transformed fertile maize plants,
said method comprising: obtaining an immature embryo from a maize
plant; delivering a nucleic acid sequence of interest by
microprojectile bombardment within 14 days to said immature embryo;
selecting for transformed cells; and, regenerating fertile
transformed slants.
2. The method of claim 1 wherein said maize plant is selected from
the genotypes CG00526, LH51, CG00708, LH82, CG00716, CG00717,
LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678,
CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661,
CG00632, CG00662, CG00712, CG00684 and CG00689.
3. The method of claim 1 wherein an immature embryo is pretreated
with an osmotically-active substance while in the presence of a
nutrient medium.
4. The method of claim 3 wherein an osmotically-active substance is
selected from sucrose, sorbitol, polyethylene glycol, glucose and
mannitol.
5. The method of claim 1 wherein said nucleic acid sequence codes
for an insecticidal protein.
6. The method of claim 5 wherein said nucleic acid sequence codes
for an insecticidal protein from the genus Bacillus.
7. The method of claim 6 wherein said nucleic acid sequence codes
for the protein endotoxin of Bacillus thurigiensis.
8. The method of claim 7 wherein said nucleic acid sequence is a
maize optimized coding sequence.
9. The method of claim 5 wherein said nucleic acid sequence is one
of the sequences recited in Tables 5 to 11 of the above
specification.
10. The method of claim 5 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
11. The method of claim 6 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
12. The method of claim 7 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
13. The method of claim 8 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
14. The method of claim 9 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase-promoter and a
root-preferred promoter.
15. The method of claim 1 wherein said nucleic acid sequence codes
for the regulatory sequences known as C1 and B-Peru which control
anthocyanin expression.
16. The method of claim 15 whereby the transformed cells are
selected visually using the expression of anthocyanin controlled by
the introduced genes.
17. A stably transformed maize plant produced by: obtaining an
immature embryo from a maize plant; delivering a nucleic acid
sequence of interest by microprojectile bombardment within 14 days
to said immature embryo; selecting for transformed cells; and,
regenerating fertile transformed plants.
18. The stably transformed maize plant of claim 17 having
essentially one of the genotypes known as CG00526, LH51, CG00708,
LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642,
CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686,
CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or
CG00689.
19. A hybrid obtained by crossing the stably transformed plant of
claim 18 with another maize plant.
20. The maize plant of claim 18 wherein said nucleic acid sequence
encodes an insecticidal protein.
21. The maize plant of claim 20 wherein said nucleic acid sequence
codes for an insecticidal protein from the genus Bacillus.
22. The maize plant of claim 21 wherein said nucleic acid sequence
codes for the protein endotoxin of Bacillus thurigiensis.
23. The maize plant of claim 18 wherein said nucleic acid sequence
is a maize optimized coding sequence.
24. The maize plant of claim 20 wherein said nucleic acid sequence
is one of the sequences recited in Tables 5 to 11 of the above
specification.
25. The maize plant of claim 20 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
26. The maize plant of claim 21 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
27. The maize plant of claim 22 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
28. The maize plant of claim 23 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
29. The maize plant of claim 24 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
30. The maize plant of claim 18 wherein said nucleic acid sequence
codes for the regulatory sequences known as C1 and B-Peru which
control anthocyanin expression.
31. The hybrid of claim 19 wherein said nucleic acid sequence
encodes an insecticidal protein.
32. The hybrid of claim 31 wherein said nucleic acid sequence codes
for an insecticidal protein from the genus Bacillus.
33. The hybrid of claim 32 wherein said nucleic acid sequence codes
for the protein endotoxin of Bacillus thurigiensis.
34. The hybrid of claim 31 wherein said nucleic acid sequence is a
maize optimized coding sequence.
35. The hybrid of claim 31 wherein said nucleic acid sequence is
one of the sequences recited in Tables 5 to 11 of the above
specification.
36. The hybrid of claim 31 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
37. The hybrid of claim 32 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
38. The hybrid of claim 33 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
39. The hybrid of claim 34 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
40. The hybrid of claim 35 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
41. The hybrid of claim 19 wherein said nucleic acid sequence codes
for the regulatory sequences known as C1 and B-Peru which control
anthocyanin expression.
42. A method for producing stably transformed fertile maize plants,
said method comprising: obtaining Type I embryogenic callus derived
from a viable portion of a maize plant; delivering a nucleic acid
sequence of interest by microprojectile bombardment to said Type I
embryogenic callus; selecting for transformed cells; and,
regenerating fertile transformed plants.
43. The method of claim 42 wherein said maize plant is selected
from the genotypes CG00526, LH51, CG00708, LH82, G00716, CG00717,
LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678,
CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661,
CG00632, CG00662, CG00712, CG00684 and CG00689.
44. The method of claim 42 wherein a Type I embryogenic callus is
pretreated with an osmotically-active substance while in the
presence of a nutrient medium.
45. The method of claim 44 wherein an osmotically-active substance
is selected from sucrose, sorbitol, polyethylene glycol, glucose,
mannitol.
46. The method of claim 42 wherein said nucleic acid sequence codes
for an insecticidal protein.
47. The method of claim 46 wherein said nucleic acid sequence codes
for an insecticidal protein from the genus Bacillus.
48. The method of claim 47 wherein said nucleic acid sequence codes
for the protein endotoxin of Bacillus thurigiensis.
49. The method of claim 46 wherein said nucleic acid sequence is a
maize optimized coding sequence.
50. The method of claim 46 wherein said nucleic acid sequence is
one of the sequences recited in Tables 5 to 11 of the above
specification.
51. The method of claim 46 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
52. The method of claim 47 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
53. The method of claim 48 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
54. The method of claim 49 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
55. The method of claim 50 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
56. The method of claim 42 wherein said nucleic acid sequence codes
for the regulatory sequences known as C1 and B-Peru which control
anthocyanin expression.
57. A stably transformed maize plant produced by: obtaining Type I
embryogenic callus derived from a viable portion of a maize plant;
delivering a nucleic acid sequence of interest by microprojectile
bombardment to said Type I embryogenic callus; selecting for
transformed cells; and, regenerating fertile transformed
plants.
58. The stably transformed maize plant of claim 57 having
essentially one of the genotypes known as CG00526, LH51, CG00708,
LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642,
CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686,
CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or
CG00689.
59. A hybrid obtained by crossing the stably transformed plant of
claim 58 with another maize plant.
60. The maize plant of claim 58 wherein said nucleic acid sequence
encodes an insecticidal protein.
61. The maize plant of claim 60 wherein said nucleic acid sequence
codes for an insecticidal protein from the genus Bacillus.
62. The maize plant of claim 61 wherein said nucleic acid sequence
codes for the protein endotoxin of Bacillus thurigiensis.
63. The maize plant of claim 58 wherein said nucleic acid sequence
is a maize optimized coding sequence.
64. The maize plant of claim 60 wherein said nucleic acid sequence
is one of the sequences recited in Tables 5 to 11 of the above
specification.
65. The maize plant of claim 60 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
66. The maize plant of claim 61 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
67. The maize plant of claim 62 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
68. The maize plant of claim 63 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
69. The maize plant of claim 64 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
70. The maize plant of claim 58 wherein said nucleic acid sequence
codes for the regulatory sequences known as C1 and B-Peru which
control anthocyanin expression.
71. The hybrid of claim 59 wherein said nucleic acid sequence
encodes an insecticidal protein.
72. The hybrid of claim 71 wherein said nucleic acid sequence codes
for an insecticidal protein from the genus Bacillus.
73. The hybrid of claim 72 wherein said nucleic acid sequence codes
for the protein endotoxin of Bacillus thurigiensis.
74. The hybrid of claim 71 wherein said nucleic acid sequence is a
maize optimized coding sequence.
75. The hybrid of claim 71 wherein said nucleic acid sequence is
one of the sequences recited in Tables 5 to 11 of the above
specification.
76. The hybrid of claim 71 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
77. The hybrid of claim 72 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
78. The hybrid of claim 73 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
79. The hybrid of claim 74 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
80. The hybrid of claim 75 wherein said nucleic acid sequence
comprises a promoter from a pith-preferred promoter, a
pollen-specific promoter, a PEP Carboxlase promoter and a
root-preferred promoter.
81. The hybrid of claim 59 wherein said nucleic acid sequence codes
for the regulatory sequences known as C1 and B-Peru which control
anthocyanin expression.
Description
[0001] This application is a continuation-in-part application of
U.S. Ser. No. 951, 715 filed Sep. 25, 1992, which is a
continuation-in-part application of U.S. Ser. No. 772, 027 filed
Oct. 4, 1991 which disclosures are herein incorporated in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the transformation of maize
genotypes by microprojectile bombardment.
BACKGROUND OF THE INVENTION
[0003] The use of genetic engineering to introduce new
agronomically important traits into maize such as insect resistance
will have many commercial benefits. In order to accomplish this in
the most expedient fashion it is necessary to have a method of
transformation that can be used with maize genotypes that are
commercially valuable.
[0004] The majority of instances of maize transformation have used
a genotype known as A188, or derivatives of A188. This is because
these lines are easily established in vitro as an embryogenic line
that forms Type II, or friable, embryogenic callus and suspension
cultures. Such Type II cultures have been exclusively preferred as
a recipient of introduced genes in transformation methods.
Unfortunately, A188 is an inferior inbred for the development of
commercially important hybrids. (Hodges et al., Biotechnology,
4:219, 1986). Working with such "model" maize lines as A188 is
disadvantageous in that extensive breeding is usually required in
order to develop maize lines with a desirable genetic composition.
What is needed is a method that can be used with commercially
valuable maize lines without the need for reliance on such "model"
systems based on Type II or suspension cultures.
[0005] Microprojectile bombardment has been advanced as an
effective transformation technique for cells, including cells of
plants. In Sanford et al., Particulate Science and Technology, 5:
27-37 (1987) it was reported that microprojectile bombardment was
effective to deliver nucleic acid into the cytoplasm of plant cells
of Allium cepa (onion). Christou et al., Plant Physiology 87:
671-674 (1988) reported the stable transformation of soybean callus
with a kanamycin resistance gene via microprojectile bombardment.
Christou et al. reported penetration at approximately 0.1 to 5% of
cells. Christou further reported observable levels of NPTII enzyme
activity and resistance in the transformed calli of up to 400 mg/L
of kanamycin. McCabe et al., Bio/Technology 6: 923-926 (1988)
report the stable transformation of Glycine max (soybean) using
microprojectile bombardment. McCabe et al. further report the
recovery of a transformed R.sub.1 plant from an R.sub.0 chimeric
plant.
[0006] Transformation of monocots and, in particular, commercially
valuable maize lines, has been problematic. Although there have
been several reports of stable plant transformation utilizing the
microprojectile bombardment technique, such transformation has not
resulted in the production of fertile, regenerated transgenic maize
plants of a commercially valuable genotype--each report used the
genotype A188 or its derivatives (Fromm et al, BioTechnology,
8:833, 1990; Walters et al., Pl. Mol. Biol. 18:189, 1992,
Gordon-Kamm et al., Plant Cell, 2:603, 1990). There are two reports
of maize transformation using commercially valuable lines but both
rely on the availability of Type II, friable embryogenic callus as
a recipient for gene delivery (Jayne et al., 1991 Meeting of the
International Society for Plant Molecular Biology, Abstract #338;
Aves et al., 1992 World Congress on Cell and Tissue Culture, In
Vitro 28:124A, Abstract #P-1134). Prior to the present invention,
successful direct transformation of commercially valuable maize
lines has not been achieved using microprojectile bombardment of
immature zygotic embryos or Type I embryogenic callus.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 shows the plasmid map of the vector pCIB3064
containing the 35S/bar chimeric gene.
[0008] FIG. 2 shows the plasmid map of the vector pCIB3089
containing the 35S/B-Peru chimeric gene.
[0009] FIG. 3 shows the plasmid map of the vector pCIB4421
containing the PEP Carboxylase promoter fused to the synthetic BT
coding sequence.
[0010] FIG. 4 shows the plasmid map of the vector pCIB4430
containing the pollen-specific promoter fused to the synthetic BT
coding sequence.
[0011] FIG. 5 shows the plasmid map of the vector pCIB4431
containing the pollen-specific promoter fused to the synthetic BT
coding sequence and the PEP Carboxylase promoter fused to the
synthetic BT coding sequence.
[0012] FIG. 6 shows the plasmid map of the vector pCIB4433
containing the pith-preferred promoter fused to the synthetic BT
coding sequence and the 35S/bar chimeric gene.
[0013] FIG. 7 shows the plasmid map of the vector pCIB4436
containing the 35S/C1 chimeric gene.
SUMMARY OF THE INVENTION
[0014] The present invention is drawn to the stable transformation
of maize with nucleic acid sequences of interest, the regeneration
of fertile transgenic maize plants and their subsequent use for the
creation of commercially valuable lines and hybrids created with
those lines. In the invention, immature zygotic embryos are
subjected to high velocity microprojectile bombardment as a means
of gene delivery within about 14 days after excision from the
plant. After initiation of embryogenic callus and selection for
transformed cells, stably transformed plants may be regenerated
which express the foreign genes of interest. Alternatively, callus
derived from immature zygotic embryos having the Type I character
may also be employed as a recipient for gene delivery. The method
is applicable to any genotype of maize, especially commercially
important ones. In this manner, the method produces transformed
maize lines of commercial importance and their hybrid
combinations.
DETAILED DESCRIPTION OF THE INVENTION
[0015] A method for the transformation of any maize line and the
regeneration of transgenic maize plants is provided. The method
involves the delivery of nucleic acids, or particularly, genes of
interest, directly to immature zygotic embryos. Alternatively, said
nucleic acids or genes of interest may be delivered to
serially-propagated Type I embryogenic callus obtained from
immature zygotic embryos. Stably transformed cells are obtained and
are regenerated into whole, fertile plants that express the foreign
gene(s). Furthermore, the fertile transformed plants are capable of
producing transformed progeny that express the foreign gene(s). The
method provides for the direct transformation of commercially
important maize genotypes, for obtaining transformed inbreds and
for their hybrid combinations.
[0016] Embryogenic callus of maize is obtained by the process of
somatic embryogenesis. Somatic embryogenesis is a process by which
fully or partially-formed embryos arise from somatic tissue. This
is in contrast to zygotic embryogenesis whereby embryos form from
gametic tissue. Somatic embryogenesis may be induced from several
viable tissues of maize, including leaf bases (Conger et al., Pl.
Cell Rep. 6:345, 1987), tassel primordia (Rhodes et al., Pl. Sci.
46:225, 1986) and immature embryos (Green et al., Crop Science
15:417, 1975). In the present invention, immature embryos are the
preferred source of embryogenic callus.
[0017] Immature zygotic embryos of maize can be obtained by
pollinating an ear with viable pollen then removing the ear from
the plant at some later time. Typically, immature embryos for use
as sources of embryogenic callus are in the size range of about 0.5
mm to about 3.0 mm, more particularly about 1.0 to about 2.5,
especially preferred from 1.5 to 2.0. The immature embryos may, for
example, be removed from the ear individually by dissection or in
bulk by "creaming" the kernels. Isolated immature embryos are
placed with the zygotic embryo axis side in contact with an
appropriate nutrient medium (Green et al., supra, 1975).
Embryogenic callus is typically observed on the immature embryo
within about 14 days of placement on the medium. This initial
period is called the "initiation" step. After the initiation step,
the embryogenic callus is generally transferred to a different
medium for establishment and maintenance of serially-propagatable
embryogenic callus (called the "maintenance" medium), although the
same medium as that for "initiation" may also be used. Incubation
of cultures typically takes place at 25 C in the dark or under low
light. Embryogenic callus can be obtained in this way from a wide
variety of maize genotypes.
[0018] Two main types of embryogenic callus have been described in
the scientific literature. Type I embryogenic callus has been
defined as "translucent, convoluted and compact callus" (Tomes et
al., Theor. Appl. Genet. 70:505-509, 1985) or as "compact,
morphologically complex" (Armstrong et al., Planta, 164:207-214
(1985)). Type II embryogenic callus has been defined as "friable
and fast growing [callus] with well defined somatic embryos with
suspensor-like structures" (Tomes et al., supra, 1985) or as
"friable, embryogenic" (Armstrong et al., supra, 1985). It is
within the scope of this invention that either or both types of
embryogenic callus may be obtained from the immature zygotic
embryos, or other viable tissue such as tassel primordia, leaf
bases or meristems which may also be used as a source of
embryogenic callus.
[0019] In order to obtain embryogenic callus the isolated immature
maize embryos must be cultured on an appropriate medium. Many types
of medium have been shown to be useful for the establishment of
embryogenic callus from a variety of genotypes, including some
commercially important ones (Hodges et al., Bio/Technology 4:219,
1986; Duncan et al., Planta 165:322, 1985). In practice, a
preferred medium must be found experimentally for each genotype.
Typically in such an experimental procedure a selection of basal
media, sucrose concentrations, and growth regulator types and
concentrations are combined in a factorial arrangement. Immature
embryos from each genotype to be tested are placed onto medium
representing each factorial combination. Initiation frequencies are
scored for each medium and the ones producing the highest scores
are used in a second round of experimentation. In this second
round, the selected media combinations are further optimized for
the individual genotypes by fine-tuning the growth-regulator type
and concentration, and sucrose concentration. For example, Table I
below indicates the preferred medium for the initiation of
embryogenic callus for several of the genotypes disclosed in this
invention.
1TABLE I Genotype Initiation Medium LH51 MS basal medium, G10
amendments, 6% sucrose, 5 mg/L dicamba CG00716 JMS basal medium, G5
amendments, 9% sucrose, 5 mg/L dicamba CG00526 D basal medium, G8
amendments, 2% sucrose, 5 mg/L chloramben CG00642 JMS basal medium,
G5 amendments, 10% sucrose, 4 mg/L dicamba LH82 LM basal medium, G6
amendments, 4% sucrose, 0.2 mg/L 2,4-D CG00689 MS basal medium, G1
amendments, 6% sucrose, 0.5 mg/L 2,4-D CGNC4206 MS basal medium, G1
amendments, 6% sucrose, 0.5 2,4-D CG00629 D basal medium, G8
amendments, 2% sucrose, 5 mg/L chloramben (H99.times.FR16) JMS
basal medium, G5 amendments, 2% sucrose, 1 xPa91 mg/l dicamba Hi II
JMS basal medium, G5 amendments, 2% sucrose, 10 mg/L silver
nitrate, 5 mg/L dicamba
[0020] The basal media formulas used for the initiation of
embryogenic callus of the genotypes in Table I may be found in the
following, citations and is herein incorporated by reference: "D",
(Duncan et al., Planta 165:322, 1985); "KM", (Kao et al., Planta
126:105, 1975); "LM", (Litvay et al., Plant Cell Reports 4:325,
1985); "MS", (Murashige et al, Physiologia Plantarum 15:473, 1962).
The basal medium "JMS" is the medium known as "SH" (Schenk et al.,
Can. J. Bot. 50:199, 1972) modified by replacing the inorganic
nitrogen compounds with those found in "MS" (Murashige et al.,
supra, 1962).
[0021] The formulas of the amendments used for the initiation of
embryogenic callus of the genotypes in Table II are found in the
following table. The amendments used with the "KM" basal medium
were as described in Kao et al., supra, 1975).
2 TABLE II Amendment Formulas, per Liter of Medium G1 G5 G6 G8 G10
Component mg Nicotinic Acid 0.5 5.0 0.5 0.2 0.5 Pyridoxine-HCl 0.5
0.5 0.1 0.2 0.5 Thiamine-HCl 0.1 5.0 0.1 0.5 0.1 Glycine 2.0 -- --
-- 2.0 myo-Inositol 100 100 100 -- 100 Choline HCl -- -- -- 0.1 --
Riboflavin -- -- -- 0.1 -- Biotin -- -- -- 0.1 -- Folic Acid -- --
-- 0.5 -- CaPantothenate -- -- -- 0.1 -- Cyanocobalamin -- -- --
0.014 -- Casein hydrolysate 100 100 100 -- -- Proline 2800 2800
2800 -- --
[0022] Often, the preferred "maintenance" medium must be determined
experimentally, as is done for "initiation". Table III describes
the "maintenance" medium found to be useful for several of the
genotypes disclosed in the present invention.
3 TABLE II Genotype Maintenance Medium LH51 MS basal medium, G10
amendments, 3% sucrose, 0.25 mg/L 2,4-D CG00716 N6 basal medium, 25
mM proline, 100 mg casein hydrolysate, 2% sucrose, 1.5 mg/L 2,4-D
CG00526 D basal medium, G8 amendments, 2% sucrose, 0.5 mg/L 2,4-D
LH82 LM basal medium, G6 amendments, 3% sucrose, 3 mg/L chloramben
CG00689 MS basal medium, G10 amendments, 2% sucrose, 1.5 mg/L 2,4-D
CGNC4206 D basal medium, G8 amendments, 2% sucrose, 1.2 2,4-D
(H99.times.FR16) D basal medium, G4 amendments, 2% sucrose, 0.5
xPa91 mg/l 2,4-D Hi II N6 basal medium, G5 amendments, 2% sucrose,
1 mg/L 2,4-D
[0023] In Table III, "N6" basal medium refers to that described in
Chu et al. Scientia Sinica, XVIII:659, 1975. The formulas for the
amendments used in "maintenance" medium may be found in Table
II.
[0024] According to the present invention, nucleic acid sequences
or genes of interest are delivered to the immature embryos within
the "initiation" step of the development of an embryogenic callus,
i.e., within about 14 days of the placement of the immature embryos
on a nutrient medium capable of supporting the intiation and
development of embryogenic callus. After gene delivery and
initiation of embryogenic callus, the embryogenic callus is
transferred to a "maintenance" medium for subculture in either the
presence or absence of a selection agent. In another embodiment of
the invention, Type I callus developed using the methods above may
also be used as a recipient of the nucleic acid sequences or genes
of interest. In this instance, after delivery of the nucleic acid
of interest, the Type I embryogenic callus is normally transferred
to fresh "maintenance" medium, with or without a selection
agent.
[0025] There are available many types of microprojectile
bombardment devices, all working on essentially the same principle
of accelerating micrometer size particles sufficient to cause
penetration into the target tissues and cells. Since it is known
that bombardment devices based on gunpowder do not work for the
present invention, the preferred devices are those that use some
form of gas expansion for the accelerating force for the
microprojectiles such as air, carbon dioxide, nitrogen, water vapor
or helium. The most preferred device for use in the claimed method
is one based on compressed helium such as the DuPont
PDS-1000/He.
[0026] For the bombardment of Type I embryogenic callus, the callus
must be subdivided into smaller pieces. This can be achieved by
chopping, maceration, dissection or other mechanical means. It is
also possible to subdivide the callus through enzymatic means.
Enzymes that digest the cell wall or cell-wall components may be
used to reduce the integrity of the callus mass. Such enzymes
include cellulases, macerases, pectinases, hemi-cellulases and
others well known in the art. After the callus is subdivided, it is
rinsed several times with liquid culture medium.
[0027] In preparation for gene delivery by microprojectile
bombardment, either the immature embryos or Type I embryogenic
callus may optionally be pre-treated with an osmotically-active
agent to plasmolyze the cells for a period from about 1 to about 24
hours, preferably from about 1 to about 12 hours, most preferably
from about I to about 6 hours. Typically, the recipient material is
treated with a concentration of sucrose that produces an osmotic
pressure in the medium that is greater than that in the recipient
material. The concentration,of sucrose may range from about 2 to
about 18%, preferably from about 6 to about 12%. It is also within
the scope of this invention that other osmotically-active agents
may be used in concentrations sufficient to cause the plasmolysis
of the cells of the recipient material, such as sorbitol, glucose,
mannitol and various molecular weight ranges of polyethylene
glycol. The recipient material is kept in the presence of the
osmotically-active agent after gene delivery for a period of about
1 to about 24 hours, preferably from about 3 to about 18 hours,
most preferably from about 10 to about 18 hours.
[0028] In a preferred embodiment of the invention, the set-up and
use of the microprojectile bombardment device, as well as the
targeting of the recipient material, is described below. Other
arrangements are also possible.
[0029] The DNA is prepared for microprojectile bombardment by
chemical precipitation in the presence of micrometer size gold,
essentially according to the published procedure of DuPont. In
addition to gold, other dense particles of micrometer size may be
used, such as tungsten or platinum. In one modification of the
DuPont procedure, the particles themselves are first prepared by
suspending them in water and sonicating. The sonicated particles
are then pelleted by centrifugation and resuspended in an aqueous
solution of 50% glycerol. Particles prepared in this way are then
aliquoted into individual tubes containing approximately 3 mg of
gold particles per tube in a volume of 50 ul. DNA is added to each
tube in varying amounts, depending upon the number of plasmids to
be used, their sizes and the final concentration of DNA desired.
For example, in a typical preparation to include four plasmids for
use in the present invention, 2 ug of pCIB3089, 2 ug pCIB4436, 3 ug
pCIB4433 and 4 ug pCIB4430 are added to each tube of aliquoted gold
particles. Next, about 50 ul of 2.5 M CaCl2 and about 20 ul of 1M
spermidine are added, in that order, to each tube while vortexing
for about 3 minutes. The DNA/gold complex is then gently
centrifuged. The supernatant is removed, the particles are washed
once with 250 ul of absolute ethanol, pelleted again and then
resuspended in about 75 ul of fresh absolute ethanol. Each tube
prepared in this way is enough of the DNA/gold complex for six
"shots" with the PDS-1000/He. Ten ul of the well-suspended DNA/gold
complex is pipetted onto the macrocarrier sheet in a vibration-free
environment.
[0030] In the PDS-1000/He device, a burst of helium is released by
rupture of a plastic disk which is available in different pressure
grades. For example, single disks, or combinations of disks, can be
obtained which rupture at 200, 450, 650, 900, 1100 1350, 1550,
1800, 2000 and 2200 pounds per square inch of helium. This burst of
gas propels the macrocarrier sheet, which is stopped by a stainless
steel screen. The screen may be of different mesh sizes, such as
10.times.10, 16.times.16, 24.times.24, etc. Other settings are the
macrocarrier flight distances, gap distance, and particle flight
distance. These settings are described in detail in the
manufacturer's user's manual. Typically, a gap distance of about
5.5 mm, a macrocarrier flight distance of about 10 mm and a
particle flight distance of about 6 to 9 cm is used. In addition, a
screen or baffle may be inserted within the particle flight
distance between the stopping screen and the target plate. Such a
screen or baffle disturbs the shock wave from the expanding gas
thereby reducing damage to the target. In one example, stainless
steel screens with an opening of about 100 um is used. Other
opening sizes and material composition may be used.
[0031] The immature embryos or Type I embryogenic callus may be
arranged on the target plate in different patterns. Through a
series of experiments, optimized patterns were developed for
immature embryos. In one optimized pattern, the immature embryos
are arranged in a circular pattern, the circle being about 2 cm in
diameter. The immature embryos are placed on the periphery of the
circle. Approximately 36 immature embryos are placed onto each
target plate. Furthermore, the target plate may be angled relative
to the microcarrier launch assembly. This ensures maximum
saturation of the basipetal portion of the immature embryo by the
particle spread. It is the basipetal portion of the immature embryo
that gives rise to the embryogenic response.
[0032] In one example of the bombardment of Type I embryogenic
callus, the callus is placed on the periphery of a circle about 1
cm diameter on a nutrient medium. The mechanical settings of the
bombardment device may be placed at positions similar or identical
to the settings recited above for the bombardment of immature
embryos.
[0033] It should be noted that the target pattern and gun settings
are interrelated. In other words, the use of other mechanical
settings on the microprojectile bombardment device can produce
other optimal arrangements of the recipient tissue on the target
plate. Other combinations of mechanical settings and target
patterns are within the scope of the invention.
[0034] The recombinant DNA molecules of the invention also can
include a marker gene to facilitate selection in recombinant plant
cells. Examples of markers include resistance to a biocide such as
an antibiotic, e.g. kanamycin, hygromycin, chloramphenicol,
paramomycin, methotrexate and bleomycin, or a herbicide such as
imidazolones, sulfonylureas, glyphosate, phosphinothricin (PPT),
glufosinate, or bialaphos. Marker genes are well known in the art.
In one embodiment of the invention the selection agent
phosphinothricin is used in conjunction with the selectable marker
gene known as bar, which encodes for the enzyme phosphinothricin
acetyltransferase. This enzyme acetylates the phosphinothricin
molecule, thereby rendering it non-toxic to plant cells. The bar
gene may be operably linked to a constitutive promoter such as the
CaMV 35S promoter; the CaMV 19S promoter; A. tumefaciens promoters
such as octopine synthase promoters, mannopine synthase promoters,
nopaline synthase promoters, or other opine synthase promoters;
ubiquitin promoters, actin promoters, histone promoters and tubulin
promoters. Other promoters may also be used. In one embodiment of
the present invention, the bar coding sequence is operably linked
to the CaMV 35S promoter.
[0035] Selection of transformed cells in vitro is accomplished by
including the selection agent of interest in the medium used to
induce and support the establishment of embryogenic callus. Only
cells in which the selectable marker is integrated into the
chromosome and is expressed, i.e., are transformed, will survive
the selection agent. Over time, the cells will grow into an
embryogenic callus in the presence of the selection agent
eventually reaching a mass sufficient for regeneration of whole,
fertile plants. Typically, such embryogenic callus can be
maintained for long periods of time in the presence of selection
agent and still retain its ability to produce whole, fertile
plants. The minimum time required to obtain an embryogenic callus
of sufficient mass under selection pressure can range from about 2
weeks to about 24 weeks, more preferably about 4 weeks to about 20
weeks, most preferably about 8 weeks to about 12 weeks.
[0036] Transformed cells may also be selected in vitro through
visual means. In order to accomplish this, a scorable marker is
generally used. Examples of scorable markers would be the
regulatory or structural genes controlling anthocyanin
biosynthesis, GUS (beta-glucuronidase), luciferase, opine
synthetases, thaumatin, beta-galactosidase, unique synthetic
epitopes designed for easy detection by ELISA, phycobiliproteins
and various fluorigenic substances. In a particular embodiment of
the present invention the use is made of coding sequences for the
anthocyanin regulatory genes known in the art as C1 and B-Peru
(Goff et al., EMBO Journal, 9: 2517, 1990). Such coding sequences,
operably linked to one or more of the several constitutive
promoters listed above, can be used to isolate transformants on the
basis of the red pigmentation of cells transformed with such
genes.
[0037] Fertile transformed plants may be regenerated from isolated
transformed embryogenic callus by several means. In general, the
transformed embryogenic callus is transferred to a nutrient medium
devoid of an auxin-type phytohormone, or is passaged through a
series of nutrient media with diminishing concentrations of
phytohormone. Other phytohormones may be used during the
regeneration step, such as cytokinins (both natural and synthetic)
and gibberellins. In some instances, inhibitors of phytohormone
action may also be used, such as silver nitrate, ancymidol or TIBA.
Other amendments to the nutrient medium for regeneration such as
activated charcoal and various gelling agents are also known in the
art.
[0038] In one example of the present invention, embryogenic callus
was removed from maintenance medium containing PPT after 12 weeks
and placed on regeneration medium containing MS basal medium, 3%
sucrose, 0.25 mg/L 2,4-D and 5 mg/L benzyladenine. After 2 weeks
the embryogenic callus began to regenerate and was transferred to
MS basal medium with 3% sucrose. Plantlets are obtained during
incubation under light. Such plants may be transferred to a
greenhouse environment after sufficient root mass has
developed.
[0039] In another embodiment of the invention, transformed
embryogenic callus is transferred from maintenance medium
containing phosphinothricin to regeneration medium consisting of MS
medium, 3% sucrose and also containing phosphinothricin.
Regeneration under such selective conditions also produces
plantlets during incubation under light, which again may be
transferred to a greenhouse environment after sufficient root mass
has developed.
[0040] As will be evident to one of skill in the art, now that a
method has been provided for the stable transformation of maize
according to the claimed method, any gene of interest can be used
in the methods of the invention. For example, a maize plant can be
engineered to express disease and insect resistance genes, genes
conferring nutritional value, genes to confer male and/or female
sterility, antifungal, antibacterial or antiviral genes, and the
like. Likewise, the method can be used to transfer any nucleic acid
to control gene expression. For example, the DNA to be transferred
could encode antisense RNA.
[0041] The present invention also encompasses the production by the
disclosed method of transformed maize plants and progeny containing
a gene or genes which encode for and express insecticidal proteins.
Such genes may be derived from the genus Bacillus, for example
Bacillus thuringiensis. In a particular embodiment of the present
invention is the use of the claimed method to produce transformed
maize plants containing a gene or genes whose nucleic acid sequence
has been altered so as to be optimized for expression in maize. A
complete description of the creation of said gene or genes may be
found in U.S. Ser. No. 951,715 which is herein incorporated by
reference. A summary of that disclosure is given below.
[0042] A nucleic acid sequence of interest in the present invention
includes one which encodes the production of an insecticidal toxin,
preferably a polypeptide sharing substantially the amino acid
sequence of an insecticidal crystal protein toxin normally produced
by Bacillus thuringiensis (BT). The synthetic gene may encode a
truncated or full-length insecticidal protein (IP) Especially
preferred are synthetic nucleic sequences which encode a
polypeptide effective against insects of the order Lepidoptera and
Coleoptera, and synthetic nucleic acid sequences which encode a
polypeptide having an amino acid sequence essentially the same as
one of the crystal protein toxins of Bacillus thuringiensis variety
kurstaki, HD-1.
[0043] The present invention provides the use of synthetic nucleic
acid sequences to yield high level expression of active
insecticidal proteins in maize plants. The synthetic,nucleic acid
sequences of the present invention have been modified to resemble a
maize gene in terms of codon usage and G+C content. As a result of
these modifications, the synthetic nucleic acid sequences of the
present invention do not contain the potential processing sites
which are present in the native gene. The resulting synthetic
nucleic acid sequences (synthetic BT IP coding sequences) and plant
transformation vectors containing this synthetic nucleic acid
sequence (synthetic BT IP genes) result in surprisingly increased
expression of the synthetic BT IP gene, compared to the native BT
IP gene, in terms of insecticidal protein production in plants,
particularly maize. The high level of expression results in maize
cells and plants that exhibit resistance to lepidopteran insects,
preferably European Corn Borer and Diatrea saccharalis, the
Sugarcane Borer.
[0044] For example, the maize codon usage table described in Murray
et al., Nucleic Acids Research, 17:477 1989, the disclosure of
which is incorporated herein by reference, was used to reverse
translate the amino acid sequence of the toxin produced by the
Bacillus thuringiensis subsp. kurstaki HD-1 cryIA(b) gene, using
only the most preferred maize codons. This sequence was
subsequently modified to eliminate unwanted restriction
endonuclease sites, and to create desired restriction endonuclease
sites. These modifications were designed to facilitate cloning of
the gene without appreciably altering the codon usage or the maize
optimized sequence. During the cloning procedure, in order to
facilitate cloning of the gene, other modifications were made in a
region that appears especially susceptible to errors induced during
cloning by the polymerase chain reaction (PCR).
[0045] In a preferred embodiment of the present invention, the
protein produced by the synthetic nucleic acid sequence of interest
is effective against insects of the order Lepidoptera or
Coleoptera. In a more preferred embodiment, the polypeptide encoded
by the synthetic nucleic acid sequence of interest consists
essentially of the full-length or a truncated amino acid sequence
of an insecticidal protein normally produced by Bacillus
thuringiensis var. kurstaki HD-1. In a particular embodiment, the
synthetic DNA sequence encodes a polypeptide consisting essentially
of a truncated amino acid sequence of the BT CryIA(b) protein.
[0046] The present invention also encompasses the use of maize
optimized coding sequences encoding other polypeptides, including
those of other Bacillus thuringiensis insecticidal polypeptides or
insecticidal proteins from other sources. For example, cryIB genes
from Bacillus thuringiensis can be maize optimized, and then stably
introduced into maize plants. It is also within the scope of this
invention that the nucleic acid sequences of interest which encode
insecticidal proteins may be either in the native or synthetic
forms, optimized for expression in maize, and derived from any
species of the genus Bacillus.
[0047] The insecticidal proteins produced by the nucleic acid
sequences of interest in the present invention are expressed in a
maize plant in an amount sufficient to control insect pests, i.e.
insect controlling amounts. It is recognized that the amount of
expression of insecticidal protein in a plant necessary to control
insects may vary depending upon species of plant, type of insect,
environmental factors and the like. Generally, the insect
population will be kept below the economic threshold which varies
from plant to plant. For example, to control European corn borer in
maize, the economic threshold is 0.5 eggmass/plant which translates
to about 10 larvae/plant.
[0048] In the present invention, the coding sequence of the nucleic
acids of interest is a synthetic maize-optimized gene under the
control of regulatory elements such as promoters which direct
expression of the coding sequence. Such regulatory elements, for
example, include monocot or maize and other monocot functional
promoters to provide expression of the gene in various parts of the
maize plant.
[0049] The regulatory element may be constitutive. That is, it may
promote continuous and stable expression of the gene. Such
promoters include but are not limited to the CaMV 35S promoter; the
CaMV 19S promoter; A. tumefaciens promoters such as octopine
synthase promoters, mannopine synthase promoters, nopaline synthase
promoters, or other opine synthase promoters; ubiquitin promoters,
actin promoters, histone promoters and tubulin promoters.
[0050] The regulatory element may also be a tissue-preferential or
tissue-specific promoter. The term "tissue-preferred promoter" is
used to indicate that a given regulatory DNA sequence will promote
a higher level of transcription of an associated structural gene or
DNA coding sequence, or of expression of the product of the
associated gene as indicated by any conventional RNA or protein
assay, or that a given DNA sequence will demonstrate some
differential effect; i.e., that the transcription of the associated
DNA sequences or the expression of a gene product is greater in
some tissue than in all other tissues of the plant. Preferably, the
tissue-preferential promoter may direct higher expression of the
synthetic gene in leaves, stems, roots and/or pollen than in seed.
"Tissue-specific promoter" is used to indicate that a given
regulatory DNA sequence will promote transcription of an associated
coding DNA sequence essentially entirely in one or more tissues of
a plant, or in one type of tissue, e.g. green tissue, while
essentially no transcription of that associated coding DNA sequence
will occur in all other tissues or types of tissues of the plant.
Numerous promoters whose expression are known to vary in a tissue
specific manner are known in the art. One such example is the maize
phosphoenol pyruvate carboxylase (PEPC), which is green
tissue-specific. See, for example, Hudspeth, R. L. and Grula, J.
W., Plant Molecular Biology 12:579-589, 1989. Other green
tissue-specific promoters include chlorophyll a/b binding protein
promoters and RubisCO small subunit promoters.
[0051] The regulatory element may also be inducible, such as by
heat stress, water stress, insect feeding or chemical induction, or
may be developmentally regulated.
[0052] In one preferred nucleic acid of interest, the regulatory
element is a pith-preferred promoter isolated from a maize TrpA
gene. That is, the promoter in its native state is operatively
associated with a maize tryptophan synthase-alpha subunit gene
(hereinafter "TrpA"). The encoded protein has a molecular mass of
about 38 kD. Together with another alpha subunit and two beta
subunits, TrpA forms a multimeric enzyme, tryptophan synthase. Each
subunit can operate separately, but they function more efficiently
together.
[0053] The nucleic acids of interest in the present invention also
include purified pollen-specific promoters obtainable from a plant
calcium-dependent phosphate kinase (CDPK) gene. That is, in its
native state, the promoter is operably linked to a plant CDPK gene.
In a preferred embodiment, the promoter is isolated from a maize
CDPK gene. By "pollen-specific," it is meant that the expression of
an operatively associated structural gene of interest is
substantially exclusively (i.e. essentially entirely) in the pollen
of a plant, and is negligible in all other plant parts. By "CDPK,"
it is meant a plant protein kinase which has a high affinity for
calcium, but not calmodulin, and requires calcium, but not
calmodulin, for its catalytic activity.
[0054] In another nucleic acid of interest, the regulatory element
is a root-preferential promoter. A complete description of such a
root promoter and the methods for finding one may be found in U.S.
Ser. No. 508,207 filed Apr. 12, 1990, the relevant parts of which
are herein incorporated by reference. Briefly, a root-preferential
promoter was isolated from a gene whose cDNA was found by
differential screening of a cDNA library from maize. A cDNA clone
so obtained was used to isolate a homologous genomic clone from
maize. The protein encoded by the isolated clone was identified as
a metallothionein-like protein.
[0055] Maize is easily hybridized because of the physical distance
between the tassel (male part) and the ear (female part). The
method of hybridization first involves the development of inbred
lines. Inbred lines are maize plants that are essentially the same
genetically from generation to generation. Inbreds are produced by
taking the pollen from one maize plant and transferring the pollen
to the silk of a receptive maize ear of that same plant. Selections
for uniformity and agronomic performance are made and the process
is repeated until the seeds from the ears of the plants produce
genetically the same plants and the line is pure. A hybrid maize
plant is produced by crossing one elite inbred maize plant with one
or more other, genetically different and diverse, inbred maize
plant. The crossing consists of taking the pollen from one inbred
elite maize plant and transferring the pollen to the silk of a
receptive ear of the other elite inbred maize plant. The seed from
crossing of two inbreds is a first generation hybrid and is called
a F1. The F1 of commercially valuable inbreds have better yields,
standability, and improvement in other important characteristics
than either of the parents. This phenomenon is called hybrid
vigor.
[0056] In the present invention, commercially-valuable inbred lines
of maize are directly transformed through the disclosed methods of
delivering nucleic acid sequences of interest to either immature
zygotic embryos obtained from such lines or Type I embryogenic
callus derived from immature zygotic embryos of such lines. The
ability to directly transform maize lines of commercial value is a
distinct advantage of the claimed invention in that the generations
of backcrossing required when the starting material is not
commercially valuable can be avoided, thereby reducing the time and
cost of commercialization. Alternatively, the present invention
also discloses the direct transformation of hybrids of inbred lines
using the claimed methods.
[0057] Many hybrid crosses have been successfully made using the
transformed, commercially-valuable plants of the claimed invention.
For example, the transformed genotype CG00526 of Example 2, below,
has been crossed to genotypes CG00689, CG00716, CG00661, CG00642,
and LH82 thereby creating hybrids possessing insecticidal
activity.
[0058] Using the methods of the present invention any hybrid
expressing a gene of interest can be created by transforming an
inbred line with the gene of interest and using such transformed
line to create the hybrid. A transformed hybrid may also be
obtained according to the present invention by directly
transforming either immature zygotic embryos obtained from said
hybrid plant or by transforming Type I embryogenic callus derived
from immature zygotic embryos obtained from said-hybrid plant.
[0059] In another embodiment of the claimed invention, it is also
possible to produce maize plants that have an altered phenotype of
anthocyanin pigmentation. This can be accomplished through the use
of the disclosed chimeric genes for the constitutive promotion of
the genes known as C1 and B-Peru. That activation of the
biosynthetic pathway for anthocyanin can be achieved in this way in
embryogenic callus was reported by Goff et al., EMBO Journal, 9:
2517-2522, 1990. In the present invention, the above said genes
were used to produce plants and progeny according to the claimed
method whose color phenotype was altered.
[0060] Commercially-valuable maize genotypes having altered color
phenotype may have benefit to the process of plant breeding. For
example, the use of the monoploid inducing gene known as id
(Kermicle, Science 166:1422-1424, 1969) can be used to create a
haploid having a paternal nuclear constitution. Monoploid inducers
creating a haploid having a maternal nuclear constitution are also
known (for example, Coe, The American Naturalist, XCIII: 381-382,
1959). Because of the low frequency of such an event, it would be
advantageous to have an easily screened color phenotype which would
allow the identification of the haploids. By using the claimed
method and genes of interest, it is possible to obtain a
transformed maize line having pigmented seeds, which can be used
with a monoploid inducing line. Haploid seed can then be identified
by either the presence or absence of seed pigmentation, depending
upon the genotypes and crossing methods used.
[0061] Since a variety of altered color phenotypes can be produced
by the present invention, examples of which are described below, it
is further envisioned that other uses in plant breeding may be
found for the claimed plants. As another example of such utility,
it is possible to link operably, molecularly, biochemically or
genetically, or any combination thereof, the expression of the
altered color phenotype with the expression of the insecticidal
activity produced by transformation of maize according to the
claimed methods. Such a link would allow rapid, visual
identification of plants within a segregating population of plants
and possesing the gene or gene products. Linkages of the altered
color phenotype and genotype to other traits of agronomic interest
are also envisioned. The ability to perform such identification
would translate into reduced costs and time for the plant
breeder.
EXAMPLES
[0062] The following examples further describe the materials and
methods used in carrying out the invention and the subsequent
results. They are offered by way of illustration, and their
recitation should not be considered as a limitation of the claimed
invention.
Example 1
Bioassay of Transformed Maize for Insecticidal Activity and
Quantitation of an Insecticidal Protein
[0063] Transformed plants were assayed for insecticidal activity
and the presence of a BT protein resulting from the expression of
the maize-optimized coding sequence of a synthetic BT gene. The
procedure is similar for any maize plant transformed with a BT gene
but is described here using as an example a cryIA(b) gene, its
expressed product, and resistance to European corn borer.
[0064] Insecticidal activity was determined by insect bioassay. One
to four 4 cm sections are cut from an extended leaf of a
transformed maize plant. Each leaf piece is placed on a moistened
filter disc in a 50.times.9 mm petri dish. Five neonate European
corn borer larvae are placed on each leaf piece. Since each plant
is sampled multiple times this makes a total of 5-20 larvae per
plant. The petri dishes are incubated at 29.5.degree. C. and leaf
feeding damage and mortality data are scored at 24, 48, and 72
hours.
[0065] Quantitative determination of a cryIA(b) IP in the leaves of
transgenic plants is performed using enzyme-linked immunosorbant
assays (ELISA) as disclosed in Clark M F, Lister R M, Bar-Joseph M:
ELISA Techniques. In: Weissbach A, Weissbach H (eds) Methods in
Enzymology 118:742-766, Academic Press, Florida (1986).
Immunoaffinity purified polyclonal rabbit and goat antibodies
specific for the B. thuringiensis subsp. kurstaki IP were used to
determine ng IP per mg soluble protein from crude extracts of leaf
samples. The sensitivity of the double sandwich ELISA is 1-5 ng IP
per mg soluble protein using 50 ug of total protein per ELISA
microtiter dish well. Corn extracts were made by grinding leaf
tissue in gauze lined plastic bags using a hand held ball-bearing
homogenizer (AGDIA, Elkart Ind.) in the presence of extraction
buffer (50 mM Na2CO3 pH 9.51 100 mM NaCl, 0.05% Triton, 0.05%
Tween, 1 mM PMSF and 1 .mu.M leupeptin). Protein determination was
performed using the Bio-Rad (Richmond, Calif.) protein assay.
Example 2
Transformation of the CG00526 Genotype of Maize by Direct
Bombarding of Immature Zygotic Embryos and Isolation of Transformed
Callus With the Use of Phosphinothricin as a Selection Agent
[0066] Immature embryos for experiment KC-65 were obtained
approximately 14 days after self-pollination. The immature zygotic
embryos were divided among different target plates containing
medium capable of inducing and supporting embryogenic callus
formation at 36 immature embryos per plate. The immature zygotic
embryos were bombarded with a mixture of the plasmids pCIB3064 and
pCIB4431 using the PDS-1000/He device from DuPont. The plasmids
were precipitated onto 1 um gold particles essentially according to
the published procedure from DuPont, as described above. Each
target plate was shot one time with the plasmid and gold
preparation. Since the plasmid pCIB3064 contained a chimeric gene
coding for resistance to phosphinothricin this substance was used
to select transformed cells in vitro. This selection was applied at
3 mg/L one day after bombardment and maintained for a total of 12
weeks. The embryogenic callus so obtained was regenerated in the
absence of the selection agent phosphinothricin. Plants were
obtained from one isolated line of embryogenic callus and given the
Event Number 176. All plants were tested by the chlorophenol red
(CR) test for resistance to PPT as described in U.S. patent
application Ser. No. 759,243, filed Sep. 13, 1991, the relevant
portions of which are hereby incorporated herein by reference. This
assay utilizes a pH sensitive indicator dye to show which cells are
growing in the presence of PPT. Cells which grow produce a pH
change in the media and turn the indicator yellow (from red).
Plants expressing the resistance gene to PPT are easily seen in
this test. Of the 38 plants regenerated for Event Number 176, eight
were positive in this test and 30 were negative. Plants positive by
the CR test were assayed by PCR for the presence of the synthetic
BT gene. Of the eight positive plants from the CR test, 5 were
positive for the presence of the synthetic BT gene, 2 were negative
and 1 died during propagation. These five remaining plants were
bioassayed and found to be resistant to European Corn Borer. DNA
was isolated from plant #11 using standard techniques and analysed
by Southern blot analysis. It was found to contain sequences which
hybridize with probes generated from the synthetic cryIA(b) gene
and with a probe generated from the PAT gene. These results showed
integration of these genes into the genome of maize. Plant #11 was
shown by ELISA to contain 2,195 ng BT protein per mg soluble
protein in the leaf tissue, consistent with the use of the
leaf-specific promoter from PEPC operably linked to a synthetic BT
gene. Plants resistant to European Corn Borer and expressing the
introduced BT gene are transformed.
Example 3
Transformation of the Hi II Genotype of Maize by Direct Bombarding
of Immature Zygotic Embryos and Isolation of Transformed Callus
Without the Use of a Selection Agent
[0067] Ear number ED42 was self-pollinated and immature zygotic
embryos were obtained approximately 10 days later. Two hundred and
eighty eight immature zygotic embryos were divided among 7
different target plates containing a medium capable of inducing and
supporting the formation of embryogenic callus. After two days, the
immature zygotic embryos were transferred to the same medium but
containing 12% sucrose. After 5 hours, the immature zygotic embryos
were bombarded with a mixture of the plasmids pCIB3089, pCIB4430,
pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The
plasmids were precipitated onto 1 um gold particles essentially
according to the published procedure from DuPont, as described
above. The particles were delivered using burst pressures of 450,
650 and 900 psi of helium. Each target plate was shot twice with
the plasmid and gold particle preparation. After overnight
incubation, the immature embryos were transferred to fresh
maintenance medium containing 2% sucrose. Since the plasmids
pCIB3089 and pCIB4436 contain the C1 and B-Peru genes which
regulate anthocyanin production, the appearance of red,
multicellular sectors on the developing embryogenic callus was used
to select and isolate transformed cells, eventually obtaining a
homogeneous callus line. Embryogenic callus was regenerated in both
the absence and presence of the selection agent phosphinothricin,
resistance to which was conferred by a chimeric gene present in
plasmid pCIB4433. Plants were obtained from a total of nine
isolated embryogenic callus lines and were given the Event Numbers
197, 198, 208, 211, 219, 255, 261, 281 and 284. Leaf tissue from
plants from each event were assayed for resistance to European Corn
Borer. Plants from Event Numbers 208 and 211 were susceptible to
European Corn Borer whereas plants from Event Numbers 197, 198,
219, 255, and 261 were resistant. All the plants that were
resistant to European Corn Borer also expressed the introduced,
leaf-specific PEPC-promoted synthetic BT gene as evidenced by the
detection of BT protein using an ELISA assay. Plants resistant to
European Corn Borer and expressing the introduced BT gene are
transformed.
Example 4
Transformation of the Hi II Genotype of Maize by Direct Bombarding
of Immature Zygotic Embryos and Isolation of Transformed Callus
With the Use of Phosphinothricin as a Selection Agent
[0068] Ear number ED47 was self-pollinated and immature zygotic
embryos were obtained approximately 10 days later. Approximately
two hundred and sixty immature zygotic embryos were divided among 8
different target plates containing a medium capable of inducing and
supporting the formation of embryogenic callus. After two days, the
immature zygotic embryos were transferred to the same medium but
containing 12% sucrose. After 5 hours, the immature zygotic embryos
were bombarded with a mixture of the plasmids pCIB3089, pCIB4430,
pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The
plasmids were precipitated onto 1 um gold particles essentially
according to the published procedure from DuPont, as described
above. The particles were delivered using a burst pressure of 900
psi of helium. Each target plate was shot twice with the plasmid
and gold particle preparation. After overnight incubation, the
immature embryos were transferred to fresh maintenance medium
containing 2% sucrose. Since the plasmid pCIB4433 contained a
chimeric gene coding for resistance to phosphinothricin this
substance was used to select transformed cells in vitro. The
selection agent was applied at 10 mg/L 14 days after gene delivery
and increased to 20-40 mg/L after approximately one month. The
embryogenic callus so obtained was regenerated in the presence of
the selection agent phosphinothricin. Plants were obtained from a
total of eleven isolated embryogenic callus lines and were given
the Event Numbers 220, 221, 222, 223, 225, 230, 231, 232, 233, 269,
274. Plants from each event were assayed for resistance to European
Corn Borer. Leaf tissue of plants from Event Numbers 220, 221, 222,
223, 225, 231 and 233 were resistant. All the plants that were
resistant to European Corn Borer also expressed the introduced,
leaf-specific PEPC-promoted synthetic BT gene as evidenced by the
detection of BT protein using an ELISA assay. Plants resistant to
European Corn Borer and expressing the introduced BT gene are
transformed. Plants from Event Numbers 230, 232, 269 and 274 were
not completely tested.
Example 5
Transformation of the CG00526 Genotype of Maize by Bombarding of
Type I Callus Derived From Immature Zygotic Embryos and Isolation
of Transformed Callus With the Use of Phosphinothricin as a
Selection Agent
[0069] Type I callus was obtained from immature zygotic embryos
using standard culture techniques. For gene delivery, approximately
300 mg of the Type I callus was prepared by chopping with a scalpel
blade, rinsing 3 times with standard culture media containing 18%
sucrose and immediately placed onto semi-solid culture medium again
containing 18% sucrose. After approximately 4 hours, the tissue was
bombarded using the PDS-1000/He Biolistic device from DuPont. The
plasmids pCIB4430 and pCIB4433 were precipitated onto 1 um gold
particles using the standard protocol from DuPont. Approximately 16
hours after gene delivery the callus was transferred to standard
culture medium containing 2% sucrose and 10 mg/L phosphinothricin
as Basta. The callus was subcultured on selection for 8 weeks,
after which surviving and growing callus was transferred to
standard regeneration medium for the production of plants.
Example 6
Transformation of the LH51 Genotype of Maize by Bombarding of Type
I Callus Derived From Immature Zygotic Embryos and Isolation of
Transformed Callus With the Use of Phosphinothricin as a Selection
Agent
[0070] Type I callus was obtained from immature zygotic embryos
using standard culture techniques. For gene delivery, approximately
300 mg of the Type I callus was prepared by chopping with a scalpel
blade, rinsing 3 times with standard culture media containing 12%
sucrose and immediately placed onto semi-solid culture medium again
containing 12% sucrose. After approximately 4 hours, the tissue was
bombarded using the PDS-1000/He Biolistic device from DuPont. The
plasmids pCIB4430 and pCIB4433 were precipitated onto 1 um gold
particles using essentially the standard protocol from DuPont as
described above. Approximately 16 hours after gene delivery the
callus was transferred to standard culture medium containing 2%
sucrose and 1 mg/L phosphinothricin as Basta. The callus was
subcultured on selection for 8 weeks, after which surviving and
growing callus was transferred to standard regeneration medium for
the production of plants.
Example 7
Transformation of the CG00526 Genotype of Maize by Direct
Bombarding of Immature Zygotic Embryos and Isolation of Transformed
Callus With the Use of Phosphinothricin as a Selection Agent
[0071] Ear numbers JS21, JS22, JS23, JS24 and JS25 were
self-pollinated and immature zygotic embryos were obtained
approximately 10 days later. Approximately eight hundred and forty
immature zygotic embryos were divided among 14 different target
plates containing a medium capable of inducing and supporting the
formation of embryogenic callus. The immature zygotic embryos were
transferred immediately to the same medium but containing 12%
sucrose. After 5 hours, the immature zygotic embryos were bombarded
with a mixture of the plasmids pCIB3089, pCIB4433, pCIB4436 using
the PDS-1000/He device from DuPont. The plasmids were precipitated
onto 1 um gold particles essentially according to the published
procedure from DuPont, as described above. The particles were
delivered using a burst pressure of 1550 psi of helium. Each target
plate was shot twice with the plasmid and gold particle
preparation. Since the plasmid pCIB4433 contained a chimeric gene
coding for resistance to phosphinothricin this substance was used
to select transformed cells in vitro. The selection agent was
applied at 10 mg/L on the day of gene delivery and increased to 40
mg/L after approximately one month. The embryogenic callus so
obtained was regenerated in the presence of the selection agent
phosphinothricin. Plants were obtained from a total of eight
isolated embryogenic callus lines and were given the Event Numbers
187, 188, 191, 192, 193, 196, 228 and 229. Plants from each event
were assayed for resistance to European Corn Borer. Plants from
Event Numbers 191 and 193 exhibited insect resistance in the pith
in accordance with the use of the pith-preferred synthetic BT
construct. All the plants that were resistant to European Corn
Borer also expressed the introduced chimeric BT gene as evidenced
by the detection of BT protein in the pith using an ELISA assay.
Plants resistant to European Corn Borer and expressing the
introduced BT gene are transformed.
Example 8
Transformation of the (H99xFR16)xPa91 Genotype of Maize by Direct
Bombarding of Immature Zygotic Embryos and Isolation of Transformed
Callus With the Use of Phosphinothricin as a Selection Agent
[0072] Ear numbers GP5 and JS26 were self-pollinated and immature
zygotic embryos were obtained approximately 10 days later.
Approximately three hundred and thirty immature zygotic embryos
were divided among 5 different target plates containing a medium
capable of inducing and supporting the formation of embryogenic
callus. After two days the immature zygotic embryos were
transferred to the same medium but containing 12% sucrose. After
approximately 5 hours, the immature zygotic embryos were bombarded
with a mixture of the plasmids pCIB3089, pCIB4430, pCIB4433,
pCIB4436 using the PDS-1000/He device from DuPont. The plasmids
were precipitated onto 1 um gold particles essentially according to
the published procedure from DuPont, as described above. The
particles were delivered using a burst pressure of 1300 psi of
helium. Each target plate was shot twice with the plasmid and gold
particle preparation. Since the plasmid pCIB4433 contained a
chimeric gene coding for resistance to phosphinothricin
this'substance was used to select transformed cells in vitro. The
selection agent was applied at 10 mg/L 3 weeks after the day of
gene delivery. The embryogenic callus so obtained was regenerated.
Plants were obtained from three isolated embryogenic callus lines
and were given the Event Numbers 242, 247 and 260. Plants from each
event were assayed for resistance to European Corn Borer. Plants
from Event Numbers 247 and 260 exhibited insect resistance
indicating that they were transformed.
Example 9
Transformation of the CG00526 Genotype of Maize by Direct
Bombarding of Immature Zygotic Embryos and Isolation of Transformed
Callus With the Use of Phosphinothricin as a Selection Agent
[0073] Immature zygotic embryos for the experiment KM-124 were
obtained approximately 14 days after self-pollination.
Approximately one hundred and five immature zygotic embryos were
divided among 4 different target plates containing a medium capable
of inducing and supporting the formation of embryogenic callus. The
immature zygotic embryos were bombarded with a mixture of the
plasmids pCIB4421 and pCIB4433 using the PDS-1000/He device from
DuPont.
[0074] The plasmids were precipitated onto 1 um gold particles
essentially according to the published procedure from DuPont, as
described above. The particles were delivered using a burst
pressure of 1550 psi of helium. Each target plate was shot once
with the plasmid and gold particle preparation. Since the plasmid
pCIB4433 contained a chimeric gene coding for resistance to
phosphinothricin this substance was used, as Basta, to select
transformed cells in vitro. The selection agent was applied at 5
mg/L one day after gene delivery and maintained for a total of 12
weeks. The embryogenic callus so obtained was regenerated in the
absence of the selection agent phosphinothricin. Plants were
obtained from one isolated embryogenic callus line and was given
the Event Number 268. Plants were assayed for resistance to
European Corn Borer. One of the 5 plants obtained is resistant to
European Corn Borer and is transformed.
Example 10
Color Phenotypes Exhibited by Plants Transformed With the C1 and
B-Peru Coding Sequences According to the Claimed Methods
[0075] Both genotypes Hi II and CG00526 were transformed with the
C1 and B-Peru chimeric genes recited above in FIGS. 2 and 7. A
variety of stably expressed altered color phenotypes were obtained,
a partial listing of which appears in Table IV, below.
4TABLE IV BT Event Number Color Phenotype 197 Red roots, red
anthers 208 Red roots 211 Red roots 213 Red stripe shoot 204 Red
anthers, pink silks 210 Red stripe shoot, red root, red anther, red
silk, red embryo 239 Red shoot, red root, red silk, normal embryo
207 Red anthers, red silks
[0076] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0077] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
* * * * *