Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Mutation employing a chemical mutagenic agent
Reexamination Certificate
2000-12-18
2004-02-24
Grunberg, Anne Marie (Department: 1661)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Mutation employing a chemical mutagenic agent
C435S444000, C435S440000, C435S447000, C435S446000, C435S445000, C435S443000, C800S276000, C800S270000, C800S260000
Reexamination Certificate
active
06696294
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to genetic mutations and in particular to methods of generating and identifying genetic mutations in polyploid plant species.
BACKGROUND OF THE INVENTION
In eukaryotic organisms, genetic information is encoded by DNA strands organized into sets of several chromosomes, or genomes, located within the cell nucleus. An organism containing a single copy of each chromosome set is referred to as a genetically monoploid organism. A convenient abbreviation for the monoploid complement of chromosomes is the letter “n”. For example, wheats have a basic monoploid complement of seven chromosomes, thus n=7. Most eukaryotic organisms, however, contain two copies of each member of the monoploid complement of chromosomes and are referred to as being genetically diploid (2n), with each chromosome existing as a member of a homologous pair of chromosomes. In some organisms, the diploid genome (i.e., the sum total of the genetic information encoded by the diploid number of chromosomes) is further duplicated to yield a chromosome complement consisting of multiple copies of the monoploid set of chromosomes.
Polyploid, is the generic term for an organism having more than the diploid number of chromosome sets, or genomes. Polyploidy is predominantly, although not exclusively, found in plants, especially within the agriculturally important cereal species, such as wheat and oats. Over the course of agricultural history, numerous polyploid varieties of crop species have evolved, possibly because of the improved vigor, larger grain or plant size often associated with polyploidy. Polyploidy may naturally arise by the spontaneous duplication of one or more genomes (autopolyploidy), or by the much more common process of genetically combining two or more genomes, or complete sets of chromosomes, from genetically different parents (allopolyploidy). For example, the spontaneous, natural doubling of the chromosome set of a diploid (2n) species, results in the creation of a novel autotetraploid (4n) species. The two diploid (2n) genomes that constitute the autotetraploid (4n) genome are referred to as homologous genomes, because they are genetically identical, having arisen by the duplication of a single diploid genome.
However, the true nature of autotetraploids is more complicated than appears, because true autoploids, whether spontaneous or artificially induced, are rarely fully fertile or genetically stable. All apparent autotetraploid species are only reproducible because their genomes have to some degree diverged, even though some pairs of genes show tetrasomic inheritance. A cross between two genetically divergent diploid (2n) species, in which reduction division during meiosis fails to occur, results in an allotetraploid (4n) species. In the case of the allotetraploid, however, the two diploid (2n) genomes that constitute the allotetraploid (4n) genome, are referred to as homoeologous genomes, because although they are genetically very similar, they are not genetically identical, having arisen by the fusion of two, comparatively different, independently evolved, diploid genomes.
The genetic information on the DNA strands of the chromosomes of all organisms is located in discrete segments of the chromosome DNA, termed genes. All genetic differences among natural (or artificial) species and varieties, results from mutational modifications in the structure and function of the genes. Such structural gene modifications in natural species and varieties are considered to have occurred spontaneously. The probable basis for such modifications is unknown, but there is evidence indicating that errors in DNA synthesis do infrequently occur, perhaps initiated by a wide variety of environmental and nutritional conditions. Mutations are important, in that they form the entire genetic basis for the evolution of species in nature and the basis for the artificial development of new plant cultivars. If enough different mutational variations are accumulated, the mutations form the basis for the development of new sub-species and species variations in all organisms, not only in plants.
In more modern times, geneticists and plant breeders have used mutation induction technology to supplement or complement the naturally-occurring genetic variations to improve numerous characteristics or properties of plants. Mutagen treatment technology has evolved over a long period of years, and only in the past 10 years or so has mutation induction, as a method to improve plants, become a technology of increasing acceptance. Although some methods for mutagen application have been described in the literature, and are useful, new techniques have been developed more recently that are significantly more effective and efficient in terms of resources. The mutagenesis technology embodied in this invention, represents an advance from the methods described in available literature (IAEA Manual on Mutation Breeding-Tech. Reports Series 119, IAEA, Vienna, 1977). Mutagenesis technologies in use for plant genetics and breeding research today, especially for small grain cereals mostly involve applications of the mutagens to seeds. The most widely used mutagens include electromagnetic radiations, X-rays and gamma rays, and nuclear radiations, such as thermal or fast neutrons, mainly because the sources of these radiations are more available. More commonly, chemical mutagens are now used in research; the preferred chemical agents are such alkylating agents as ethyl methanesulfonate (EMS), and diethyl sulfate (DES). In addition, azide in the form of sodium or potassium azide is now widely used. Less commonly employed for mutation induction are the more hazardous carcinogenic agents, such as N-methyl nitrosourea and N-ethyl nitrosourea, and the highly carcinogenic nitrogen mustard, 2-chloroethyl-dimethylamine. The nitrosoureas are especially active mutagens (
Maluszinski, M
. Acta. Soc. Bot. Pol. 51:429-440, 1982) whereas use of the nitrogen mustards poses a significant health risk to the user because these compounds are highly toxic to humans. More recently, as cell (microspore) and tissue culture research have evolved, attention is being given to the application of mutagens to accelerate the frequency of mutations regenerable from such cultures. However, mutagenesis technology in cultures is still in its developmental infancy, as are applications of the technology for plant improvement.
Most applications of mutagens to produce useful variants in crop plant species have had a primary goal, such as reducing plant height, reducing grain shattering, or changing the photoperiod response, traits for which the genetic basis in the mutagenized variety was previously unknown. Even so, unexpected, as well as expected results have been achieved from many studies. As a result, many new cultivars of crop plants have been developed via the direct release of a genetic line differing from the original genotype by an induced mutation (Konzak, C. F., Role of Induced Mutations, 1983, pp. 216-292. In: Crop Breeding-a Contemporary Basis. P. B. Vose and S. G. Blixt (eds). Pergamon Press, Oxford and New York; Micke, A. and Donini, B., 1987, Tropic. Agric. (Trinidad) 64:259-277). An even larger number of new cultivars of crop plants have been developed using induced mutations as new genetic variability, demonstrating that induced mutations not only can be useful, but also that they can be used in breeding to advance the potential yield, quality or disease resistance, of many crops (Micke, A. and Donini, B., 1987, Tropic. Agric. (Trinidad) 64:259-277). However, there remains a need for a method of inducing a wide range of mutations that is generally applicable to crop plants. Typically, mutagens have been applied to seeds of various species to induce mutations that might be expected to occur, based on an expectation that such genetic variation should be inducible. In most of the examples described in the literature, the actual numbers of mutations of a general phenotype generally have been sufficiently high for other scientists t
Christensen O'Connor Johnson & Kindness PLLC
Grunberg Anne Marie
Northwest Plant Breeding Co.
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