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
1997-03-21
2001-01-16
Hutzell, Paula K. (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, C435S468000, C435S254110, C427S004000, C424S093200, C424S093200, C424S093210, C424S093500
Reexamination Certificate
active
06175059
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This application incorporates by reference in its entirety U.S. patent application Ser. No. 08/299,608 now U.S. Pat. No. 5,614,186, inventor Charles M. Rush.
This invention relates to the treatment of plants to reduce the plant's susceptibility to severe disease caused by virulent viral pathogens. Plants inoculated with a non-virulent or mildly virulent virus do not express severe disease symptoms caused by the virulent virus even when co-inoculated with the virulent virus. The novel inoculation method includes application of viruliferous fungus to seeds by coating seeds with survival structures of the viruliferous fungus, such as cystosori of Polymyxa species.
In addition, the present invention relates to a method for delivering viruses, both wildtype and recombinant, to plants. Soilborne fungi containing recombinant, and/or wildtype, viruses are used to infect plants and thereby deliver recombinant and/or wildtype viruses to plants. In an embodiment of the present invention survival structures, such as cystosori, of soilborne fungi containing recombinant or wildtype viruses are used to coat seeds of plants susceptible to soilborne fungi such that plants growing from these seeds become infected with the recombinant or wildtype virus containing fungi thereby delivering the virus to the plant.
In addition, the present invention relates to a method for delivering foreign genes or viruses to plant cells. Specifically, the present invention relates to a method of introducing foreign genes or viruses into plant cells in planta, via a seed treatment method utilizing recombinant furoviruses and their natural fungal vectors in order to introduce specific agronomic traits, and production of desirable products such as pharmaceuticals.
2. General Background
Applied biotechnology in agriculture has been moving at an astonishing pace since the successful transformation of tobacco via Agrobacterium (Ti plasmid) was reported in 1983 (32). Over the last ten years, transformation of major crop species with foreign genes in order to introduce desirable agronomic traits has been a primary research focus in the scientific community. Today, new varieties have been developed from all the major crop species in which transgenic plants have been produced (65) and many of them are now available to farmers and consumers (87). The traits that have been or soon will be incorporated into crops include herbicide tolerance (Roundup, Basta, Buctril, Atrazine) insect (
Bacillus thuringiensis
endotoxin) and disease (chitinase, virus coat protein) resistance, quality improvement (high-stearic-acid oilseed rape, pre-colored cotton, and high methionine maize seed), and high-value biopharmaceutical products (alkaloids and vaccines) (24,87).
The methods for delivery of a single gene or set of genes to plant cells are known as transformation and transfection. These methods have the goal of incorporation of the alien gene(s) into plant cells or integration into the plant genome. This permits the expression of the foreign genes transiently or permanently. Transient expression describes a result of attempted transformation in which expression of the gene (production of mRNA) is time-limited, usually to a period of a few days or weeks. Reduction of expression may or may not be accompanied by loss of the gene itself. In contrast, permanent expression is accompanied by the transmission of the expressing foreign gene to subsequent generations.
Transfection is a process in which viral genes are transferred into plant cells through normal or modified virus activity. The viral genome may be modified to allow transfer of non-viral genes that replicate episomally in the cytoplasm or integrate into the plant genome (91). Transformation describes any of several other methods, both biological and physical, for the transfer of genes which are stably integrated into the genome. Such experiments sometimes result in transient expression of the gene. These methods include, but are not limited to, tissue-culture-dependent DNA delivery systems, such as Ti-plasmid (72, 76), PEG (97), Ca
2+
-mediated DNA uptake through protoplasts (70), liposome encapsidation, electroporation (65), microinjection (30), silicon carbide fibers (57), particle bombardment (59), as well as other plasmid-mediated gene transfers, and tissue-culture-independent systems, such as DNA injection into ovaries (23), DNA delivery along the pollen tube (69), and DNA uptake through seed imbibition (92, 105). A necessary feature of gene delivery systems is the establishment of marker genes (NPT II, CAT, BAR, HYG, GUS and LUC) (65), promoters (NOS, CaMV 35S, Ubi 1, Adh 1, Act 1 and pEMU) and introns which, in combination with the promoter regions, facilitate the selection of transgenic plants and the expression of the foreign genes (17). In general, for all the reproducible gene transfer procedures, there are biological limitations associated with their applications, such as host specificity, genotype specificity for tissue-culture-regeneration capacity, somaclonal variation, fertility loss, insufficient level of gene expression, instability of transgenes, and potential for transfer of foreign genes into weed species (65). Most processes are highly technical, expensive, lengthy and difficult to control. At present, for dicot crops, the most efficient and widely-applicable transformation method is the Agrobacterium vector system. For monocot crops, particle bombardment of shoot tips or immature embryos is the current method of choice, even though successful Agrobacterium-mediated transformation of maize and rice have been reported (49, 52). Both procedures limit the number of genotypes that can be manipulated, dependent on their tissue-culture-regeneration capacity. Below is a more detailed description of these currently available gene transfer methods along with their limitations.
Agrobacterium-Mediated Transformation
Agrobacterium gene delivery has become almost routine in many laboratories but transformation of some species using this technology has been unsuccessful. Agrobacterium causes crown gall disease in many dicot plants in nature by transferring a defined segment of DNA from its tumor-inducing (Ti) plasmid into the nuclear genome of cells through a wound area of the plants (58). This unique plant-microbial interaction has been combined with the high totipotency of many dicot leaf cells and developed into a simple leaf-disc transformation system in which gene transfer, selection and regeneration are coupled together in an efficient process (50). Tobacco is an excellent host for Agrobacterium. It responds very well in in vitro culture and was the first plant species transformed using Agrobacterium. Advances in tissue-culture regeneration techniques and effective interaction with
A. tumefaciens
have provided a reproducible method for transfer of cloned, engineered genes in many dicot crop species including tomato, lettuce, sunflower, rape, cotton, and soybean (51). Recently, Agrobacterium-mediated transformation of so-called non-host crop plants has been accomplished in maize and rice (49, 52).
There are a number of technical issues that have required attention in the application of this technique to species other than model species (e.g. tobacco). One problem is inefficiency or ineffectiveness of the selectable marker. Escapes or false positives often occur, perhaps due to loss of DNA during plant development or due to the cross-protection of wild-type cells by nearby transformed cells. This problem generally has been solved by applying selection during more susceptible growth stages or using dual selection. Some plant species or genotypes respond differently to the same selection agent even though they are transformed with the same marker gene. Therefore, new marker genes and selection agents have been developed to use in those crops. Another problem has been the interaction between Agrobacterium and the explant, sometimes limiting the survival or vigor of the explants which, in turn, reduces the ove
Fulbright & Jaworski L.L.P.
Hutzell Paula K.
Texas A & M University
Zaghmout Ousama M-Faiz
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