Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
2000-06-30
2002-03-05
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C435S419000, C435S469000, C800S298000
Reexamination Certificate
active
06353155
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improved, simplified method for transforming plants using a bacteria known as Agrobacterium.
BACKGROUND OF THE INVENTION
Recent advances in genetic engineering have provided plant breeders and geneticists with the tools to insert or transform new genetic material, usually in the form of DNA (deoxyribonucleic acid) fragments, into a plant in order to produce new kinds of plants known as transgenic plants. Such transgenic plants or crops can have unique characteristics or traits, including resistance to plant diseases, resistance to herbicides, resistance to insects, enhanced stability or shelf-life of the ultimate consumer product obtained from the plant and/or improvements in the nutritional value in the edible portions of the plant. In agriculture, a bacterium known as
Agrobacterium tumefaciens
was discovered to have the property of being able to naturally infect a host plant, and insert and integrate a portion of its DNA into the host plant's DNA. Since then, this DNA transfer mechanism has been harnessed a way to produce genetically engineered transgenic plants.
Recent scientific publications such as Bechtold et al., Methods Mol. Biol. 82:259-266 (1998); Clough et al., Plant J, 16(6):735-743 (1998); and Ye et al., Plant J., 19(3): 249-257 (August 1999) teach lengthy methods for in planta transformation of
Agrobacterium thaliana.
In these methods, a suspension of Agrobacterium is initially grown. Then the suspension is transferred to a centrifugation bottle and the bacterial cells are pelleted by spinning the centrifugation bottles on a centrifugation machine. The pelleted Agrobacteria are resuspended in a media for transforming a plant. Finally, the plants are contacted with the resuspended media for the transformation process. Although effective, such methods have the disadvantage of being time consuming and requiring expensive centrifuges to prepare the Agrobacterium. Such disadvantages are become multiplied for applications of high-throughput transformation of plants using many different types of genes or DNA. Thus, in order to prepare transgenic plants or seeds, a new approach was sought to transform plants with Agrobacterium that would be more efficient and less costly than previous methods.
SUMMARY OF THE INVENTION
In one embodiment, the present invention is directed towards a method for preparing a transgenic plant or seed comprising:
a) growing a suspension of Agrobacterium until the optical density of the suspension is about 2 to about 2.4 at a wavelength of 600 nanometers, wherein said Agrobacterium contains at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said suspension with an aqueous medium so that the optical density of the suspension is reduced to about 0.6 to about 1.5; and
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally; cultivating said treated plant to produce seed; and
e) optionally; growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.
In a second embodiment, the present invention is directed toward a method for preparing a transgenic plant or seed comprising:
a) growing a suspension of Agrobacterium until growth of Agrobacterium in the suspension is substantially completed, wherein said Agrobacterium contains at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said suspension with an aqueous medium to reduce the concentration of Agrobacterium and any other components in the growth medium and allow the Agrobacterium to infect the plant without harming it;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.
In a third embodiment, the present invention is directed toward a method for preparing a transgenic plant or seed comprising:
a) growing a suspension of Agrobacterium until growth of Agrobacterium in the suspension is substantially completed, wherein said Agrobacterium contains at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said suspension with about 2 to about 10 volumes aqueous medium per volume of suspension;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.
Preferably, the Agrobacterium is
Agrobacterium tumefaciens.
Also preferred is that the plant is Arabidopsis. Also preferred is that the aqueous medium contains a surfactant, sugar or both.
The present invention has the advantage of providing a method for transforming plants that requires significantly less time and personnel to perform compared with other known methods.
Another advantage of the present invention is that it eliminates the need to use expensive centrifuges to prepare the Agrobacterium cultures.
Still yet another advantage is that it provides a method for high-throughput transformation of plants, such as
Arabidopsis thaliana,
using many different types of DNA sequences.
REFERENCES:
Clough et al, “Flip dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana”, 1998, The Plant Journal, vol. 16(6), pp. 735-743.*
Bechtold et al., In Planta Agrobacterium-Mediated Transformation of Adult Arabidopsis thaliana Plants by Vacuum Infiltration, Methods in Molecular Biology, 82:259-266 (1998).
Ye et al., Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation, The Plant Journal, 19(3):249-257 (Aug. 1999).
Klöti Andreas S.
Mulpuri Rao
Collins Cynthia
Majka Joseph T.
McElwain Elizabeth F.
Nowak Henry P.
Paradigm Genetics, Inc.
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