Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-11-03
2004-11-23
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S294000, C435S412000, C435S424000, C435S430100, C435S419000, C435S469000
Reexamination Certificate
active
06822144
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for plant tissue culture and plant regeneration and in particular this invention relates to methods for transforming maize using
Agrobacterium.
BACKGROUND OF THE INVENTION
Agrobacterium
-mediated transformation methods have been used principally in dicotyledonous plants.
Agrobacterium
-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition,
Agrobacterium
-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
Monocotyledons are not a natural host of
Agrobacterium
. Although
Agrobacterium
-mediated transformation has been reported for asparagus (Bytebier B., et al.
Proc. Natl. Acad Sci.
USA 84:5354-5349, 1987) and for
Dioscore bublifera
(Schafer et al.
Nature
327:529-532, 1987), it was generally believed that plants in the family Gramineae could not be transformed with
Agrobacterium
(Potrykus I.
Biotechnology
8:535-543, 1990).
Grimsley et al. (
Nature
325:177-179, 1987) reported that cDNA from maize streak virus could be delivered to maize plants by
Agrobacterium tumefaciens
and that the plants became infected with the virus. The research did not demonstrate that the cDNA reached the maize genome nor did it demonstrate stable integration of streak virus nucleic acid. Later studies demonstrated that
Agrobacterium
could be used to deliver a kanamycin-resistance gene and a GUS (&bgr;-glucuronidase) gene to shoot apices of maize after shoot apex injury (Gould J. et al.
Plant Physiol.
95:426-434, 1991 and U.S. Pat. No. 5,177,010 to Goldman et al.). In these studies plants generated from the tissue exposed to
Agrobacterium
contained both transformed cells and, non-transformed cells suggesting that the method did not uniformly deliver nucleic acid to the maize tissue.
European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocotyledons using
Agrobacterium.
In this method, plant tissues were obtained from the monocotyledon maize and the tissues were exposed to
Agrobacterium
during the tissue dedifferentiation process. Hiei et al. disclose a maize transformation protocol using maize calli. Saito et al. disclose a method for transforming monocotyledons using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al. also disclose a method specific for transforming maize by exposing immature embryos to
A. tumefaciens
(
Nature Biotechnology,
1996, 14:745-750). The methods were optimized for inbred A188 maize lines. Transformation frequencies ranged from 12% to 30% at their highest for immature embryos from A188 lines that were 1.0-1.2 mm in length. Maize lines derived from crosses of A188 had significantly lower transformation frequencies ranging from 0.4% to about 5.3%. The transformation frequencies using A188 and A188 crosses are summarized in Table 1. A188 is not generally considered a commercially useful line and Ishida et al. failed to obtain recovery of stable transformants in lines other than those containing A188.
A need still exists for a method that will: (a) produce significantly higher transformation frequencies in lines other than those reported by Ishida et al. (supra); and, (b) produce transformed inbred lines other than line A188; including transformed inbreds representing a range of genetic diversities and having significant commercial utility.
SUMMARY OF THE INVENTION
This invention relates to methods for optimizing
Agrobacterium
-mediated transformation in maize. Significantly higher transformation frequencies for genotypes such as the product of A188 crossed to other inbreds would result in a higher throughput for production of transformed plants. This increased frequency would be useful, for example, to evaluate the efficacy of a larger number of genes in transgenic plants of corn or to generate a larger number of transgenic plants containing a particular foreign gene in a given period of time. Similarly, methods permitting the transformation of a variety of inbred lines would be commercially valuable.
In one aspect of this invention, the invention relates to a method for transforming maize using
Agrobacterium
comprising the steps of: contacting at least one immature embryo from a maize plant with
Agrobacterium
capable of transferring at least one gene to the embryo; co-cultivating the embryo with
Agrobacterium
; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of
Agrobacterium
, and a selective agent to select for embryos expressing the gene; and regenerating maize plants expressing the gene. In one embodiment the contacting step additionally comprises the step of contacting the immature embryos with
Agrobacterium
in a medium comprising N6 salts and in another embodiment the contacting step additionally comprises contacting the immature embryos with
Agrobacterium
in a medium comprising MS salts. Preferably the contacting step takes place in the absence of AgNO
3
. In one embodiment the embryos are cultured in a PHI basic media system and in another embodiment the embryos are cultured in a PHI combined media system. The immature embryos used in the method are preferably about 0.3 mm to about 4 mm in length and more preferably about 0.8 mm to about 2.0 mm in length. The
Agrobacterium
concentration used in the contacting step is preferably about 1×10
8
cfu/ml to about 1.5×10
9
cfu/ml and more preferably about 0.5×10
9
to about 1.0×10
9
cfu/ml. The contacting step preferably takes place in a liquid suspension and the co-cultivation step preferably takes place on a solid medium. Preferably, a medium containing MS salts is used in the regeneration step. In a preferred embodiment of this invention the method includes a resting step that comprises culturing the embryos in medium containing an antibiotic capable of inhibiting the growth of
Agrobacterium
. Preferably the embryos are cultured for about 1 to about 15 days. In one embodiment the antibiotic used is carbenicillin and a preferred concentration of carbenicillin is about 50 mg/l to about 250 mg/l. This method also relates to maize plants transformed by this method and to maize cells transformed by this method.
In another aspect of this invention, the invention relates to a method for transforming maize using
Agrobacterium
comprising the steps of: contacting at least one immature embryo from a maize plant with
Agrobacterium
capable of transferring at least one gene to said embryo in a medium comprising N6 salts; co-cultivating the embryo with
Agrobacterium
in a medium comprising N6 salts; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of
Agrobacterium
, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene in a medium comprising MS salts. Preferably, the medium of the contacting step lacks AgNO
3
and the medium of the co-cultivating step includes AgNO
3
. Preferably the
Agrobacterium
concentration used in the contacting step is about 1×10
8
cfu/ml to about 1.5×10
9
cfu/ml. Preferably, the contacting step takes place in a liquid and the co-cultivating and culturing steps take place on a solid medium. In one embodiment of this method, the method additionally comprising the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of
Agrobacterium.
Preferably the antibiotic is carbenicillin. This invention also relates to maize plants and to maize cells transformed by this method.
In yet another aspect of this invention, a method is disclosed for transforming maize using
Agrobacterium
comprising the steps of: contactin
Cai Tishu
Gu Weining
Pierce Dorothy A.
Zhao Zuo-Yu
Fox David T.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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