Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
2000-06-06
2003-12-23
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C435S468000, C435S469000, C800S278000, C800S306000
Reexamination Certificate
active
06667428
ABSTRACT:
BACKGROUND OF THE INVENTION
Genetic transformation provides a useful tool for the study of gene regulation. In addition, randomly transformed plants can be screened for valuable characteristics, such as increased number or size of commercially valuable plant organs (e.g., the fruit). Agrobacterium-mediated delivery of genetic constructs into plant genomes has proven to be a highly useful vehicle for plant cell transformation and subsequent generation of transgenic plants. However, many plants appear to be resistant to Agrobacterium-mediated delivery. For these recalcitrant plants, as well as permissive plants, methods of increasing the efficiency of transformation using Agrobacterium would be of value.
SUMMARY OF THE INVENTION
The invention is based on the unexpected discovery that cell populations with a nuclear DNA phase or content of 4C are especially susceptible to Agrobacterium-mediated gene delivery, while cells with a nuclear DNA content of greater than 4C are less susceptible to Agrbbacterium-mediated gene delivery. Thus, by selecting or enriching cell populations for 4C cells, the efficiency of plant cell transformation can be increased.
Accordingly, the invention features a method of introducing a nucleic acid into plant cells by enriching a plant cell population for cells having a nuclear DNA phase of 4C, and contacting the enriched plant cell population with a bacterium of the genus Agrobacterium to form a mixed culture, the bacterium having a T-DNA which includes the nucleic acid. The invention further includes a method of introducing a nucleic acid into plant cells by selecting, from a plurality of plant cell populations, a plant cell population containing the highest proportion of cells having a nuclear DNA phase of 4C; and contacting the selected plant cell population with a bacterium of the genus Agrobacterium to form a mixed culture, the bacterium having a T-DNA which includes the nucleic acid.
The enriched or selected plant cell population does not have to be precultured, i.e., the cells harvested from a plant are not cultured, though they can be stored, before being contacted by the bacterium. The increase in transformation efficiency can be further augmented by subjecting the mixed culture to vacuum pressure (e.g., 0.1 bar to 0.8 bar of pressure) or by removing cells having a nuclear DNA phase of greater than 4C from the plant cell population. The plant cell population can be of a particular plant tissue, such as broccoli plant tissue, tissue of a peduncle, or a seedling tissue (e.g., of a hypocotyl or a cotyledon). In addition, the T-DNA can further include a kanamycin resistance gene, and the method can further include, after the contacting step, selecting for plant cells in a medium containing kanamycin (e.g., about 25 mg/l or more, 50 to 100 mg/l, or 75 mg/l kanamycin).
The invention also features a method of producing a transgenic broccoli plant, the method comprising introducing a nucleic acid into a broccoli plant cell using the methods described above, the nucleic acid having a nucleotide sequence encoding an isopentyl transferase.
As used herein, the nuclear DNA phase refers to the nuclear DNA content as a multiple of the amount of DNA expected to be present in a haploid nuclei. A nuclear DNA phase of 4C means that the nucleus of a cell contains an amount of DNA about four times the haploid amount.
The methods of the invention are useful for high efficiency transformation of plant cells, from which transgenic plants can subsequently be generated. The methods are particularly useful for transforming plant cells known to be resistant to Agrobacterium-mediated gene delivery.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.
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Chen Long-Fang O.
Huang Jia-Yuan
Yang Shang-Fa
Academia Sinica
Fish & Richardson P.C.
Fox David T.
Kruse David H
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