Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1995-11-09
2003-08-26
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S283000, C800S286000, C800S294000
Reexamination Certificate
active
06610909
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the development of techniques for the commercial production of transgenic plants.
Background of the Invention
The genetic manipulation of plants is centuries old, and modern crop yields and disease- and pest-resistances often owe much to traditional plant genetic engineering. Classical plant breeding methods are time-consuming and subject to chance, however, so the recent advent of recombinant DNA techniques is promising. This promise is encouraging especially with respect to enabling plant geneticists to identify and to clone specific genes for desirable traits, and to introduce such genes into already useful varieties of plants.
Translating genetic engineering theory into practice, however, and then furthermore into a commercially practical reality, requires ingenuity. Gene transplantation in plants has already been accomplished at this writing—and examples are cited below—but heretofore no practical method for the commercial production of transgenic plants has been perfected.
Apart from the transgenic plant technology per se, it is known to propagate plants by replicating plant cells in culture, or “tissue culture.” An early motivating force in the development of tissue culture was the desire to improve upon the relatively slow and low yields of vegetative propagation with the quick and exponential proliferation of new plants from cell culture. Tissue culture methods are made possible by the plant physiological phenomenon of callus formation. When a plant is wounded, a patch of soft cells called a calli grows over the wound and, with time, phenolic compounds accumulate in the soft cells and harden, effectively sealing the wound. While hardened callus is the plant equivalent of scar tissue, callus is different from mammalian scar tissue with respect to its regenerative properties. If a piece of young, still-soft callus is removed and placed in a culture medium containing salts, sugars, vitamins, amino acids and the appropriate plant growth hormones, rather than harden, the cells will continue to divide and give rise to a disorganized mass of undifferentiated cells called a “callus culture.” Plant or seedling “explants,” or tissue samples, will likewise grow into similar cell cultures. The cultured cells can further be induced to redifferentiate into shoots, roots or whole plants by further culturing with the necessary hormones and growth media.
One of the most serious drawbacks with tissue culture propagation techniques has been the morphologic variation from generation to generation, a problem which is particularly notable in certain species and varieties. For example, as reported in Cassells, A. C., and Carney, B. F., “Adventitious regeneration in
Pelargonium x domesticum
Bailey,”
Acta Horticulturae,
212(II), 419-425 (1987), in stem and petiole tissue cultures of Grand Slam (as an example of
P. domesticum,
also known as Regal Pelargoniums or “Martha Washington” geraniums), up to 16% of the adventitious regenerants were variants, depending on the explant origin. The authors concluded that genome instability in Grand Slam and presumably other
P. domesticum
varieties may produce useful variation but mitigates against the use of adventitious regeneration in micropropagation.
The findings of Cassells et al. are consistent with the earlier work of Skirvin, R. M. and Janick, Jules, “Tissue Culture-Induced Variation in Scented Pelargonium ssp.,”
J. Amer. Soc. Hort. Sci.,
101(3), 281-290 (1976). Skirvin et al. compared tissue culture propagated Pelargonium plants (from root cuttings, petiole cuttings or calliclones) with plants derived from vegetative propagation, i.e., stem cuttings. The plants derived from stem cuttings were all uniform and identical to the parental clone, whereas those from the root cuttings, petiole cuttings or calliclones were all morphologically distinct with the degree of variability depending upon the cultivar. The authors conclude that the variability associated with calliclones derived from tissue culture is a pool on which selection can be imposed, implying conversely that tissue culturing of this type is inappropriate for use in attempting reliable regeneration of
Pelargonium x domesticum
varieties.
Other varieties and species, besides
Pelargonium x domesticum,
are known and/or believed to suffer morphologic variation when propagated using tissue culture. It can be easily appreciated that any substantial morphologic variation in propagation is unacceptable for commercial propagation of a desired variety or species. Thus, tissue culture methods are not always acceptable for commercial use, even with the potentially much larger yields achievable as compared with prior art vegetative propagation techniques.
Apart from tissue culture considerations, gene transplantation in plants has achieved some success at this writing. Gene introduction is generally accomplished with a vector such as Agrobacterium. As this technology developed, it was noted that Crown Gall tumors of plants arose at the site of infection of some species of the bacterium Agrobacterium. The cells of Crown Galls acquire the properties of independent, unregulated growth. In culture, such transformed cells grow in the absence of the plant hormones usually necessary for plant cell growth, and the cells retain the transformed phenotype even in the absence of the bacterium. The tumor-inducing agent in Agrobacterium is a plasmid that integrates some of its DNA into the chromosome of the host plant cells. Ti (tumor-inducing) plasmids exist in Agrobacterium cells as independently replicating genetic units.
Ti plasmids are maintained in Agrobacterium because part of the plasmid DNA, the T-DNA, carries the genes coding for the synthesis of amino acids called opines. The infected plant cell is induced to synthesize these amino acids, but the plant cannot use these amino acids. The Ti plasmid is believed to carry genes coding for enzymes that can degrade opines. Thus, Ti plasmids both make and degrade opines, within the plant cell, which the plant cell cannot metabolically use—presumably giving a selective advantage to the Agrobacterium at least with respect to utilization of the opine metabolites. A second set of genes in T-DNA codes for enzymes which lead to production of hormones which, in turn, cause the infected plant cell to divide in an unregulated way.
In summary terms, T-DNA enters a plant cell by what amounts to the equivalent of bacterial conjugation between the Agrobacterium and the plant cell. In other words, an Agrobacterium organism and a plant cell transfer their DNA in a process analogous to mating. Ultimately, T-DNA becomes incorporated into the genomic plant cell DNA in the plant cell nucleus.
All of the above background illustrates how Agrobacterium species can serve well as vectors for genetic transformation of plant cells. Early gene transfer using Ti plasmids, T-DNA and Agrobacterium was accomplished by the cointegration method, in which T-DNA was first cloned into a standard
E. coli
cloning vector, and the plant gene was subsequently cloned into a second cloning site carried by the vector. This intermediate vector was introduced into Agrobacterium organisms containing intact Ti plasmids. Recombination occurred between the homologous regions of the intermediate vector and the wild-type Ti plasmid, and on infection of a plant with the Agrobacterium the recombinant plasmid is transferred to the plant cells.
Despite the early use of the cointegration method described above—and certainly it still works—the standard method for T-DNA transfer as of this writing is called the “binary system.” The binary system was devised when investigators realized that the essential functions for transfer are supplied separately by the T-DNA itself and by the Ti plasmid, and that the components can be carried on separate vectors. The binary vector contains the borders of the T-DNA—needed for excision and integration—and the hormone-producing region of the original T-DNA can be removed and replaced with the foreign gene sequence intended
Arteca Jeannette
Arteca Richard N.
Oglevee-O'Donovan Wendy
Stoots Eleanor
Nelson Amy J.
Penn State Research Foundation
Webb Ziesenheim Bruening Logsdon Orkin & Hanson, P.C.
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