Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-04-19
2004-10-19
Kubelik, Anne (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S282000, C800S288000
Reexamination Certificate
active
06806399
ABSTRACT:
1. BACKGROUND OF THE INVENTION
The present invention relates to methods for plant genetic transformation and for products thereof. More specifically, the present invention relates to the genetic transformation of any plant species with sexual reproduction based on a pollination-fecundation process. According to the present invention, pollen grains are pre-treated with silicon carbide fibers and the transforming DNA. The present invention also involves pollinating recipient plants with pollen grains carrying the transforming DNA.
Advances in molecular biology have dramatically expanded man's ability to manipulate the germplasm of animals and plants. Genes controlling specific phenotypes, for example specific polypeptides that lend antibiotic or herbicide resistance, have been located within certain germplasm and isolated from it. Even more important has been the ability to take the genes which have been isolated from one organism and to introduce them into another organism. This transformation may be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene (heterologous transformation).
Genetic engineering of plants, which entails the isolation and manipulation of genetic material (usually in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant or plant cells, offers considerable promise to modern agriculture and plant breeding. Increased crop food values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance, the production of pharmaceuticals, chemicals and biological molecules as well as other beneficial traits are all potentially achievable through genetic engineering techniques. Once a gene has been identified, cloned, and engineered, it is still necessary to introduce it into a plant of interest in such a manner that the resulting plant is both fertile and capable of passing the gene on to its progeny.
Developments in agrobiotechnology have resulted in a tremendous expansion of the capabilities for the genetic engineering of plants. Many transgenic dicotyledonous plant species have been obtained. However, many species of plants, especially those belonging to the Monocotyledonae and particularly the Gramineae, including economically important species such as corn, wheat and rice, have proved to be very recalcitrant to stable genetic transformation. Difficulties have been encountered in achieving both: a) integrative transformation of monocot plant cells with DNA (i.e., the stable insertion of DNA into the nuclear genome of the monocot plant cells) and b) regeneration from transformed cells of phenotypically normal monocot plants, such as phenotypically normal, fertile adult monocot plants. It has been suggested that such difficulties have been predominantly due to the nonavailability of monocot cells that are competent with respect to: 1) DNA uptake, 2) integrative transformation with the taken-up DNA, and 3) regeneration of phenotypically normal, monocot plants from the transformed cells (Potrykus (1990) Bio/Technology 9:535).
Thus, the introduction of exogenous DNA into monocotyledonous species and subsequent regeneration of transformed plants has proven much more difficult than transformation and regeneration in dicotyledonous plants. Moreover, reports of methods for the transformation of monocotyledons such as maize, and subsequent production of fertile maize plants, have not been forthcoming. Consequently, success has not been achieved in this area and commercial implementation of transformation by production of fertile transgenic plants has not been achieved. Thus there is a particularly great need for methods for improving genetic characteristics. Problems in the development of genetically transformed monocotyledonous species have arisen in many general areas. For example, there is generally a lack of methods, which allow one to introduce nucleic acids into cells and yet permit efficient cell culture and eventual regeneration of fertile plants.
Genetic engineering techniques have been successfully applied principally in dicotyledonous species. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester, E. W., et al, (1984). Am. Rev. Plant Physiol 35: 387-413), polyethylene glycol (PEG) mediated DNA uptake (Lorz H., Baker B., Schell J. (1985). Mol Gen Genet 199:178-182.), electroporation of protoplasts (Fromm M. E., Taylor L. P., Walbot V. (1986). Nature 312:791-793.) and microprojectile bombardment (Klein T. M., Kornstein L., Sanford J. C., Fromm M. E. (1987). Nature 327: 70-73.).
The Agrobacterium transformation system is among the recombinant DNA technologies for genetic manipulation of plant genotypes. Virulent strains of the soil bacterium
Agrobacterium turnefaciens
are known to infect dicotyledonous plants and to elicit a neoplastic response in these plants. The tumor-inducing agent in the bacterium is a plasmid that functions by transferring some of its DNA into its host plant's cells where it is integrated into the chromosomes of the host plant's cells. This plasmid is called the Ti plasmid, and the virulence of the various strains of
A. tumefaciens
is determined in part by the vir region of the Ti plasmid which is responsible for mobilization and transfer of the T-DNA (Schell, J., Science, 237: 1176-1183 (1987)). The T-DNA section is delimited by two 23-base-pair repeats designated right border and left border, respectively. Any genetic information placed between these two border sequences may be mobilized and delivered to a susceptible host. Once incorporated into a chromosome, the T-DNA genes behave like normal dominant plant genes. They are stably maintained, expressed and sexually transmitted by transformed plants, and they are inherited in normal Mendelian fashion.
There are two common ways to transform plant cells with Agrobacterium: cultivation of Agrobacterium with cultured isolated protoplasts, or transformation of intact cells or tissues with Agrobacterium. The first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. The second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
However, the Agrobacterium transformation system, as stated, is restricted to certain dicot crops. For the majority of monocots, especially cereals (graminae) and grasses, A tumefaciens mediated gene transfer is not possible. Thus, the most important cultivated plants are not accessible for effective gene transfer.
A second frequently used process for transformation of plants is DNA direct delivery. One form of direct DNA delivery is direct gene transfer into protoplasts (using polyethyleneglycol treatment and/or electroporation). Protoplasts for use in such direct gene transfer methods have most often been obtained from embryogenic cell suspension cultures (Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6, Academic press, p. 291; OziasAkins and Lorz (1984) Trends in Biotechnology 2: 1 19). However, the success of such methods has been limited due to the fact that regeneration of phenotypically normal plants from protoplasts has been difficult to achieve for most genotypes. For example, while regeneration of fertile corn plants from protoplasts has been reported, these reported methods have been limited
Fahima Tzion
Korol Abraham
Nevo Eviator
Browdy and Neimark , P.L.L.C.
Carmel-Haifa University Economic Corporation Ltd.
Kubelik Anne
LandOfFree
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