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
1997-02-07
2001-03-06
Hutzell, Paula (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
C800S298000, C800S295000, C800S320100
Reexamination Certificate
active
06198025
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of molecular biology and genetic engineering. More specifically, the invention provides methods of introducing foreign genetic material (mRNA) into plants.
INCORPORATION BY REFERENCE
The following publications are referenced in this application by numerals in parenthesis:
1. Niu, M. C., L. C. Niu, C. Ma, Z. P. Lin and Y. L. Zhang (1980). Genetic manipulation in higher organisms. III. Detection of soy protein in seeds derived from soy mRNA-treated rice.
Scientia Sinica,
23: 119-122.
2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bactiophage T
4
. Nature,
227: 681-685.
3. Vaessen, R. T. M. J., J. Kreke and G. S. P. Groot (1981). Protein transfer to nitrocellulose filters. A simple method for quantitation of simple proteins in complex mixtures. FEBS letter 124: 193-196.
4. Mishra, N. C., M. C. Niu and E. L. Tatum (1975). Induction by RNA of inositol independence in
Neurospora crassa. Proc. Nat. Acad. Sci.,
72: 642-645.
5. Szkukalek, A., And F. Solymosy (1994). Molecular characterization of two tomato U6RNA pseudogenes generated by RNA-mediated mechanisms.
Plant Science
99:183-187.
Each of these publications is incorporated herein by reference; numbers in the text referring to these publications correspond to the numbers set forth above.
BACKGROUND OF THE INVENTION
Methods of improving various crops are known. Some of these methods involve introducing foreign, i.e. nonendogenous, DNA into plant cells or tissues and analyzing the resulting plants for presence of exogenous nucleic acid and expression of the foreign gene. Although successful in certain instances, these methods are laborious, time consuming and variable in their reproducibility. There exists a need for the further development of methods for the beneficial genetic transformation of agricultural crops to increase protein yields or otherwise improve crops for human and/or animal consumption.
SUMMARY OF THE INVENTION
In one of its aspects, this invention provides methods for producing transgenic plants by introduction therein of foreign mRNA, to impart to the resulting transgenic plants capability of synthesizing the foreign protein in subsequent generations. Based on the transfer of genetic information via mRNA molecules, as opposed to DNA molecules, in one of its aspects the invention provides a distinct improvement over methods presently employed in the art. Endogenous proteins of plants are beneficially augmented with valuable proteins from foreign sources utilizing the methods of this invention.
In another of its aspects, this invention embraces a transgenic plant expressing beneficial exogenous proteins produced by obtaining a sample of mRNA encoding the exogenous protein, incubating and inoculating seed of the plant with the mRNA under conditions whereby the mRNA enters the seed, germinating the seed and growing the transgenic plant from the seed. Desirably the sample of mRNA is obtained from soybeans, most desirably from soybean cotyledons or sprouts. Further desirably, the plant is corn and most desirably corn strain 27-1 or 85089.
In another of its aspects, this invention embraces a transgenic corn plant expressing soy globulin protein. Most desirably in this aspect of the invention the corn is strain 27-1; alternatively the corn may be strain 85089.
In a preferred practice of the invention, mRNA from soy cotyledon is isolated and purified and used for microinjection of corn seed from corn strain 27-1. The treated seeds are then germinated and resulting plants assayed for exogenous protein expression.
Following microinjection and transformation, protein presence and content in propagated transgenic corn plants is analyzed by Ouchterlony immunodiffusion assays sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The presence of soy DNA in the transformed corn is confirmed by Southern blotting using soy globulin specific radiolabeled DNA probes.
In alternative embodiments of the invention, mRNA may be isolated from soy sprouts. Additionally, other strains of corn may be utilized, including corn strain 85089. The mRNA may be microinjected into recipient seeds once or twice or even more times. The methods described result in successful production of transgenic corn plants expressing soy globulin protein, as set forth in Table 1, below.
REFERENCES:
Potrykus. Ann. Review of Plant Physiol. 1991. vol. 42: 205-225.
Napoli et al. The Plant Cell. 1989. vol. 2: 278-289.
Carvalaho et al. The EMBO Journal. 1992. vol. 11: 5995-5602.
Plant Cell Tissue Organ Culture. 1995. vol. 40: 1-15.
Topfer et al. Plant Cell. 1989. Jan. issue. vol. 1: 133-139.
Gallie et al. Plant Cell Reports. 1993. vol. 13: 119-122.
Kagawa and Hirano. 1989. Nucleic Acid Research. 1989. vol. 17: 886.
Feldmann and Marks. Molecular and General Genetics. 1987. vol. 208: 1-9.
Ausubel et al.Short Protocols in Molecular Biology. 1989.
Hutzell Paula
Quinn Charles N.
Zaghmout Ousama
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