Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1999-07-09
2001-07-17
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C800S278000, C800S301000, C536S023600, C435S200000, C435S209000, C435S320100, C435S419000
Reexamination Certificate
active
06262342
ABSTRACT:
FIELD OF THE INVENTION
One aspect of the present invention relates to the chemical regulation of gene expression. In particular, this aspect relates to non-coding DNA sequences which, in the presence of chemical regulators, regulate the transcription of associated DNA sequences in plants. Another aspect of the invention relates to DNA molecules encoding proteins capable of conferring plant disease and/or plant pest resistance. Both aspects of the invention relate, in part, to genes associated with the response of plants to pathogens.
BACKGROUND OF THE INVENTION
Advances in recombinant DNA technology coupled with advances in plant transformation and regeneration technology have made it possible to introduce new genetic material into plant cells, plants or plant tissue, thus introducing new traits, e.g., phenotypes, that enhance the value of the plant or plant tissue. Recent demonstrations of genetically engineered plants resistant to pathogens (EP-A 240 332 and EP-A 223 452) or insects (Vaeck, M. et al.,
Nature
328: 33 (1987)) and the production of herbicide tolerant plants (DeBlock, M. et al.,
EMBO J
. 6: 2513 (1987)) highlight the potential for crop improvement. The target crops can range from trees and shrubs to ornamental flowers and field crops. Indeed, it is clear that the “crop” can also be a culture of plant tissue grown in a bioreactor as a source for some natural product.
A. General Overview of Plant Transformation Technology
Various methods are known in the art to accomplish the genetic transformation of plants and plant tissues (i.e., the stable introduction of foreign DNA into plants). These include transformation by Agrobacterium species and transformation by direct gene transfer.
1. Agrobacterium-Medicated Transformations
A. tumefaciens
is the etiologic agent of crown gall, a disease of a wide range of dicotyledons and gymnosperms, that results in the formation of tumors or galls in plant tissue at the site of infection. Agrobacterium, which normally infects the plant at wound sites, carries a large extrachromosomal element called the Ti (tumor-inducing) plasmid.
Ti plasmids contain two regions required for tumorigenicity. One region is the T-DNA (transferred-DNA) which is the DNA sequence that is ultimately found stably transferred to plant genomic DNA. The other region rquired for tumorigenicity is the vir (virulence) region which has been implicated in the transfer mechanism. Although the vir region is absolutely required for stable transformation, the vir DNA is not actually transferred to the infected plant. Transformation of plant cells mediated by infection with
Agrobacterium tumefaciens
and subsequent transfer of the T-DNA alone have been well documented. See, for example, Bevan, M. W. and Chilton, M-D.,
Int. Rev. Genet
. 16: 357 (1982).
After several years of intense research in many laboratories, the Agobacterium system has been developed to permit routine transformation of a variety of plant tissue. Representative species trnmsformed in this manner include tobacco, tomato, sunflower, cotton, rapeseed, potato, soybean, and poplar. While the host range for Ti plasmid transformaton using
A. tumefaciens
as the infecting agent is known to be very large, tobacco has been a host of choice in laboratory experiments because of its ease of manipulation.
Agrobacterium rhizogenes
has also been used as a vector for plant transformation. This bacterium, which incites hairy root formation in many dicotyledonous plant species, carries a large extrachromosomal element called an Ri (root-inducing) plasmid which functions in a manner analogous to the Ti plasmid of
A. tumefaciens
. Transformation using
A. rhizogenes
has developed analogously to that of
A. tumefaciens
and has been successfully utilized to transform, for example, alfalfa,
Solanum nigrum L
., and poplar.
2. Direct Gene Transfer
Several so-called direct gene transfer procedures have been developed to transform plants and plant tissues without the use of an Agrobacterium intermediate (see, for example, Koziel et al.,
Biotechnology
11: 194-200 (1993); U.S. application Ser. No. 08/008,374, filed Jan. 25, 1993, herein incorporated by reference in its entirety). In the direct transformation of protoplasts the uptake of exogenous genetic material into a protoplast may be enhanced by use of a chemical agent or electric field. The exogenous material may then be integrated into the nuclear genome. The early work was conducted in the dicot tobacco where it was shown that the foreign DNA was incorporated and transmitted to progeny plants, see e.g. Paszkowski, J. et al.,
EMBO J
. 3: 2717 (1984); and Potrykus, I. et al.,
Mol. Gen. Genet
. 199: 169 (1985).
Monocot protoplasts have also been transformed by this procedure in, for example,
Triticum monococcum, Lolium multiflorum
(Italian ryegrass), maize, and Black Mexican sweet corn.
Alternatively exogenous DNA can be introduced into cells or protoplasts by microinjection. A solution of plasmid DNA is injected directy into the cell with a finely pulled glass needle. In this manner alfalfa protoplasts have been transformed by a variety of plasmids, see e.g. Reich, T. J. et al.,
Bio/Technology
4: 1001 (1986).
A more recently developed procedure for direct gene transfer involves bombardment of cells by microprojectiles carrying DNA, see Klein, T. M. et al.,
Nature
327: 70 (1987). In this procedure tungsten particles coated with the exogenous DNA are accelerated toward the target cells, resulting in at least transient expression in the example reported (onion).
B. Regeneration of Transformed Plant Tissue
Just as there is a variety of methods for the transformation of plant tissue, there is a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. In recent years it has become possible to regenerate many species of plants from callus tissue derived from plant explants. The plants which can be regenerated from callus include monocots, such as corn, rice, barley, wheat and rye, and dicots, such as sunflower, soybean, cotton, rapeseed and tobacco.
Regeneration of plants from tissue transformed with
A. tumefaciens
has been demonstrated for several species of plants. These include sunflower, tomato, white clover, rapeseed, cotton, tobacco, and poplar. The regeneration of alfalfa from tissue transformed with
A. rhizogenes
has also been demonstrated. Plant regeneration from protoplasts is a particularly useful technique, see Evans, D. A. et al., in: “Handbook of Plant Cell Culture”, Vol. 1, MacMillan Publ. Co., 1983, p. 124. When a plant species can be regenerated from protoplasts, then direct gene transfer procedures can be utilized, and transformation is not dependent on the use of
A. tumefaciens
. Regeneration of plants from protoplasts has been demonstrated for rice, tobacco, rapeseed, potato, eggplant, cucumber, poplar, and corn.
Various plant tissues may be utilized for transformation with foreign DNA. For instance, cotyledon shoot cultures of tomato have been utilized for Agrobacterium mediated transformation and regeneration of plants (see European application 0249432). Further examples include Brassica species (see WO 87/07299) and woody plant species, particularly poplar (see U.S. Pat. No. 4,795,855, incorporated by reference herein in its entirety).
The technological advances in plant transformation and regeneration technology highlight the potential for crop improvement via genetic engineering. There have been reports of genetically engineered tobacco and tomato plants which are resistant to infections of, for example, tobacco mosaic virus (TMV) and resistant to different classes of herbicides. Insect resistance has been engineered in tobacco and tomato plants.
C. Cell Cultures
The potential for genetic engineering is not limited to field crops but includes improvements in ornamentals, forage crops and trees. A less obvious goal for plant biotechnology, which includes both genetic engineering and tissu
Hofsteenge Jan
Meins, Jr. Frederick
Ryals John A.
Shinshi Hideaki
Sperisen Christoph
Fox David T.
Meigs J. Timothy
Novartis Finance Corporation
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