WO1999065292A1 - Procedes pour generer et identifier des plantes polypoides mutantes et utilisation desdites plantes - Google Patents

Procedes pour generer et identifier des plantes polypoides mutantes et utilisation desdites plantes Download PDF

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WO1999065292A1
WO1999065292A1 PCT/US1999/013801 US9913801W WO9965292A1 WO 1999065292 A1 WO1999065292 A1 WO 1999065292A1 US 9913801 W US9913801 W US 9913801W WO 9965292 A1 WO9965292 A1 WO 9965292A1
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seeds
target gene
plant
polyploid plant
mutants
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PCT/US1999/013801
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Calvin F. Konzak
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Konzak Calvin F
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Priority to US09/719,880 priority Critical patent/US6696294B1/en
Priority to AU49563/99A priority patent/AU4956399A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor

Definitions

  • This invention relates to genetic mutations and in particular to methods of generating and identifying genetic mutations in polyploid plant species.
  • the diploid genome i.e., the sum total of the genetic information encoded by the diploid number of chromosomes
  • 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).
  • 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.
  • 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.
  • genes 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.
  • 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.
  • chemical mutagens are now used in research; the preferred chemical agents are such alkylating agents as ethyl methanesulfonate (EMS), and diethyl sulfate (DES).
  • EMS ethyl methanesulfonate
  • DES diethyl sulfate
  • azide in the form of sodium or potassium azide is now widely used.
  • 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.
  • 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.
  • mutagenesis technology in cultures is still in its developmental infancy, as are applications of the technology for plant improvement.
  • 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.
  • the actual numbers of mutations of a general phenotype generally have been sufficiently high for other scientists to recognize them as being induced, especially the relatively common mutations of semi-dwarf, or reduced height phenotypes. But, even among these more frequent types of mutations, those at the same gene locus have been rarely isolated in the same experiment.
  • the primary evidence that the new phenotypic/genotypic variants are induced mutations has largely been assumed because of the simultaneous recovery of many other mutant phenotypes in the same study.
  • Novel mutations might convey for example, increased resistance to drought or cold, reduced plant height, non-shattering of grain, resistance to preharvest sprouting, as well as new, or modified quality characteristics, offering new market use opportunities, or might result in higher crop yields. Further, it is desirable to generate numerous mutations within a plant species in order to obtain novel phenotypes, which can be intercrossed to develop novel plant cultivars having defined, more desirable characteristics of economic value.
  • the present invention provides methods for generating and identifying mutations in any target gene of a polyploid plant species.
  • a plant is selected that has at least one pair of functional, target genes located exclusively in only one of its homoeologous, or homologous, genomes. Seed derived from the selected plant are then contacted with an effective amount of at least one mutagenic agent, the treated seed are germinated and the seeds or plants derived therefrom, are screened for mutations in the target gene.
  • the selected plant is a cereal crop plant and the mutagenic agent is a sequentially applied combination of ethyl methane sulfonate followed by sodium azide.
  • selective matings are made to construct plant genotypes with a functional target gene pair exclusively in only one of their homoeologous, or homologous, genomes. Seed derived from the constructed plants are then contacted with an effective amount of at least one mutagenic agent, the treated seed are germinated and the seed or plants derived therefrom, are screened for mutations in the target gene.
  • the constructed plant is a cereal crop plant and the mutagenic agent is a sequentially-applied combination of ethyl methane sulfonate followed by sodium azide.
  • polyploid wheat plants mutated in accordance with the methods of the present invention, are provided that include mutations in all copies of the waxy gene and so synthesize starch that has a reduced amount of, or completely lacks, amylose.
  • inventive concepts set forth herein can be used to create, select and identify mutations in any target gene of any suitable polyploid plant.
  • the mutations generated in accordance with the present invention provide a source of numerous, readily-identifiable mutations that can, if so desired, be used as germplasm to generate novel new plant cultivars, or the novel induced mutant alleles in different genomes of the polyploid, can intercrossed to generate novel phenotypes having predetermined, desirable properties.
  • FIGURE 1 graphically illustrates the distribution of polymer size of starch molecules derived from waxy wheat mutant 18 (closed diamonds) and from waxy wheat mutant 21 (open circles).
  • the x-axis represents the normalized peak area that is indicative of the amount of starch molecules having a degree of polymerization that is indicated by the y-axis.
  • FIGURE 2 graphically illustrates the degree of polymerization of starch molecules derived from waxy wheat mutant 22 derived from Kanto 107 as set forth in EXAMPLES 1 and 3.
  • the x-axis represents the normalized peak area that is indicative of the amount of starch molecules having a degree of polymerization that is indicated by the y-axis.
  • FIGURE 3 graphically illustrates the degree of polymerization of starch molecules derived from waxy wheat mutant 4 derived from Kanto 107 as set forth in EXAMPLES 1 and 3.
  • the x-axis represents the normalized peak area that is indicative of the amount of starch molecules having a degree of polymerization that is indicated by the y-axis.
  • inventive concepts set forth herein can be used to create, select, and identify mutations in any target gene of any suitable polyploid plant, thereby providing a source of numerous, readily-identifiable mutations that can, if so desired, be used as germplasm to generate novel new plant cultivars. Additionally, the novel induced mutant alleles in different genomes of the polyploid, can be intercrossed to generate novel phenotypes having predetermined, desirable properties.
  • the present invention is directed to methods for producing mutants of a target gene in a polyploid plant by: constructing a polyploid plant having at least one functional copy of a target gene located exclusively in only one of the homoeologous or homologous genomes of said polyploid plant; contacting seeds derived from the constructed polyploid plant with an effective amount of at least one mutagenic agent; germinating the mutagenized seeds; and assaying seed (or other plant tissues and/or organs) from plants derived from the germinated, mutagenized seeds to identify mutants of the target gene.
  • the present invention is directed to polyploid plants, containing a mutation in a target gene, produced by the foregoing methods.
  • the present invention is directed to seeds derived from polyploid plants, containing a mutation in a target gene, produced by the foregoing methods.
  • polyploid refers to organisms having more than the diploid (2n) number of chromosome sets, or genomes.
  • autopolyploid or “autopolyploidy” refer to polyploid organisms in which the polyploidy arose by the duplication of one or more sets of chromosomes, or genomes.
  • allopolyploid or “allopolyploidy” refer to polyploid organisms in which the polyploidy arose by genetically combining two or more complete sets of chromosomes (genomes) from genetically different parents.
  • the term "monoploid” refers to the minimum number of chromosomes that contain all of an organism's genetic information.
  • the monoploid complement of chromosomes is represented by the letter “n”.
  • the term “diploid” refers to twice the monoploid set of chromosomes, i.e.,
  • tetraploid refers to four times the monoploid set of chromosomes, i.e., 4n chromosomes.
  • hexaploid refers to six times the monoploid set of chromosomes, i.e., 6n chromosomes.
  • genomic refers to all the genetic information possessed by one monoploid set of chromosomes, and an individual organism may be genetically constructed of several pairs of genomes.
  • homologous genome refers to the like duplicate genomes of an autopolyploid organism.
  • homoeologous genome refers to the similar genomes of an allopolyploid organism.
  • genetictype refers to the genetic makeup of an organism.
  • phenotype refers to the physical trait or traits, associated with a particular gene.
  • phenotype can also be used collectively to refer to the sum of all the traits that characterize an organism.
  • locus or “genetic locus” refer to the physical location of a particular gene on a chromosome.
  • isoform refers to different forms of a protein encoded by related forms or alleles of a gene located at the same or at different loci as, for example, the different forms of the granule-bound starch synthase protein (GBSS) encoded by the gene located at the three waxy loci of hexaploid wheat. Or the different enzyme proteins encoded by the different mutant alleles of each of the waxy loci, in each of the genomes.
  • the mutants described in EXAMPLES 1-3 herein all represent alterations of the waxy gene locus, hence they may control different isoforms of the GBSS enzyme protein, if any enzyme is actually produced
  • the methods of the present invention utilize polyploid plants that have at least one pair of functional, target genes located exclusively in only one of their homoeologous, or homologous, genomes
  • an allohexaploid species useful in the practice of the present invention will have null or recessive mutations in all copies of the target gene in two of its three pairs of homoeologous genomes.
  • the remaining pair of target genes in the third homoeologous genome must be functional.
  • an allotetraploid species useful in the practice of the present invention will have null or recessive mutations in one pair of the target genes in one of the two homoeologous genomes.
  • the two copies of the target gene in the second homoeologous genome will be functional, and usually dominant.
  • Plants having null mutant gene pairs in one or more of the homoeologous genomes can be identified by means of techniques well known to those skilled in the art.
  • standard protein analytical techniques such as sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be used, by which proteins are separated on the basis of size.
  • SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
  • two-dimensional SDS-PAGE by which proteins are first separated on the basis of charge, and then separated on the basis of size, may be utilized to identify the isoforms of a target protein encoded by the cognate genes located on each of the homoeologous genomes.
  • Varieties of the plant species of interest can then be screened, using an appropriate analytical method, such as SDS-PAGE, to identify those varieties that lack specific isoforms, or which carry null mutants in one, or more, of the homoeologous genomes.
  • Protein analytical techniques, including SDS-PAGE, useful in the practice of the present invention to identify polyploid plants that lack one or more isoforms of a target protein are discussed in Homes, B.D. and Rickwood, D., (eds) 1990. Gel Electrophoresis of Proteins- A Practical Approach. Oxford Univ. Press; Scopes, R.K. and Smith, J.A., 1997. Analysis of Proteins. In Current Protocols in Molecular Biology. John Wiley & Sons.
  • Nakamura et al. Japanese Journal of Breeding, 42:681-685, 1992
  • SDS-PAGE SDS-PAGE to analyze over 1,800 wheat accessions from various parts of the world to identify genetic sources in which one or another of the homoeologous GBSS genes was inactive or deleted.
  • Nakamura et al. Japanese Journal of Breeding, 42:681-685, 1992
  • the foregoing analytical methods for identifying null mutations in one or more homoeologous loci of an allopolyploid plant, can also be applied to autopolyploid plant species, provided that the duplicated genomes have had sufficient time to diverge and accumulate sufficient mutations so that the gene products of either of the homologous loci bearing the target gene can be distinguished.
  • This cross will yield plants with A and D genome waxy null loci, but with the normal, dominant B genome non-waxy locus, thereby setting up the genotype for inducing waxy mutations in the B genome according to the practice of the present invention; (2) by crossing cv Klasic or ID377s, two wheats carrying the B genome waxy null loci, with Bai Huo, some F2 generation recombinants can be recovered which carry waxy null loci in both the B and D genomes, but with the dominant gene for the GBSS protein in the A-Wx locus. Seeds of these recombinants can be mutagenized according to the practice of the present invention to induce waxy null gene mutants in the A genome.
  • null mutants can be found among the tetraploid wheats, or if the screening system could identity a tetraploid or hexaploid wheat genotype which already has one or two of the homoeologous genomes, respectively, carrying a null mutation.
  • the subject plant/plant line can be mutagenized by contacting the plants with an effective amount of one or more mutagenic agents.
  • seeds from the subject plant line will be contacted with one or more mutagens.
  • Numerous mutagenic agents are well known to those of ordinary skill in the art (IAEA Manual on Mutation Breeding-Tech. Reports Series 119, IAEA, Vienna, 1977).
  • Radiation or beams of accelerated atomic particles can be used as mutagens. For example, gamma rays and fast neutron bombardment have been used to induce mutations in wheat.
  • chemical mutagens are utilized in the practice of the present invention, because of their ready availability and ease of application.
  • Examples of chemical mutagens useful in the practice of the present invention include, but are not limited to, ethyl methanesulfonate (EMS); diethyl sulfate (DES) and sodium azide (AZ).
  • the chemical mutagens used in the practice of the present invention are a sequential combination of EMS or DES, and sodium azide.
  • the types of mutations that are induced by mutagenic agents useful in the practice of the present invention include point mutations, deletions, inversions and substitutions of individual DNA base pairs, or of one or more DNA segments containing numerous DNA base pairs.
  • the preferred method for chemically mutating the single homoeologous, or homologous, genome bearing the sole functional pair of the target genes includes sequential applications of chemical mutagens, such as ethyl methanesulfonate or diethyl sulfate, followed by a treatment with sodium azide (AZ).
  • chemical mutagens such as ethyl methanesulfonate or diethyl sulfate
  • AZ sodium azide
  • the treatments are preferably applied to seeds that have been presoaked for 4-6 hours at room temperature (20-23°C) in either distilled water or tap water.
  • the applications of the mutagens are preferably made in distilled water solutions, but tap water can be used provided that the tap water does not contain undue amounts of metals, especially, copper contaminants (IAEA Manual on Mutation Breeding-Tech. Reports Series 119, IAEA, Vienna, 1977).
  • the EMS is applied at a concentration of 0.25-0.35 milliliters (ml) per liter distilled water for 2-2.5 hours.
  • DES can be used at 1-2 ml per liter of distilled water for a treatment time of approximately 2 hours, prior to the AZ treatment.
  • the EMS treatments are preferably followed by a treatment with lxlO -3 to 2xl0" 3 M AZ in lxl 0" 3 M phosphate buffer (pH 3.0). No wash between treatments is necessary, but the EMS solution is poured off after the selected treatment time, and before the buffer is added prior to introducing the AZ from a concentrated stock solution.
  • the AZ solution is poured off, and the seeds are rinsed with distilled water before laying them out on screen trays for redrying. Redrying should preferably not exceed 24 hours before the seeds are planted to start germination, or are placed in a refrigerator at 2-6°C. When stored in a cool refrigerator, the treated seeds can be held for several months before planting, without an undue increase in mutagen-induced injury. There is less risk of delayed injury to seeds mutagenized with DES, because the reaction rate of DES is about 9x that of EMS with water (IAEA Manual on Mutation Breeding-Tech. Reports Series 119, IAEA, Vienna, 1977).
  • the seeds are germinated, the germinated plants (Ml) are allowed to self-fertilize and M2 seed is harvested.
  • the M2 seed can be assayed for the loss of activity of the target gene if, as is the case with the waxy trait, the mutation is a property characteristic of the seed. Additionally, any tissue, organ or material derived from any plant derived from the mutagenized seed can be assayed for the partial or complete loss of activity of the target gene.
  • a target gene be identified in a particular tissue of the plant, and that an assay for a target gene product activity is available to identify those derivatives of mutagenized seed or plants that carry mutations of the target gene
  • a suitable target gene is the gene located at the waxy locus, an endosperm trait, identified in several cereal crop species including wheat and oats.
  • the protein encoded by the gene located at the waxy locus is involved in the biosynthesis of amylose starch from amylopectin starch, via debranching or linearizing the initially- synthesized amylopectin molecules, a process which is of considerable importance to the food industry.
  • Starch from cereals especially, consists mainly of two types of carbohydrate polymers; amylose, which is essentially linear, and amylopectin which is a highly branched carbohydrate polymer.
  • amylose which is essentially linear
  • amylopectin which is a highly branched carbohydrate polymer.
  • the starch granule-bound starch synthase enzyme controls the process of debranching the initially-formed amylopectin starch, leading to the formation of non-branched amylose starch.
  • a deviant, partially functional GBSS enzyme might less-effectively debranch the amylopectins, or have an unusual mode of action, resulting in the production of amylopectin starch molecules with different relative proportions of highly and less branched polymers.
  • amylose content of starch typically ranges from about 11-37% of total starch.
  • the occurrence of a deletion or null mutation in fewer than all of the genomes of a polyploid species may reduce the amount of amylose synthesized, but enough amylose will still be formed by the remaining active GBSS gene to prevent detection of the change, except by methods for identifying the enzyme itself, rather than the product, or via a more exact analysis of the starch composition by a chemical method.
  • waxy mutants that reduce, or completely abolish, the synthesis of amylose, is based on the observation that the adsorption of iodine by amylopectin and by amylose is greatly different.
  • waxy mutants can be readily distinguished by applying an iodine solution, such as IKI (iodine + potassium iodide), to the cut surface of seed endosperm and observing the color of the product. Waxy mutants are easily identified because non-mutant starch stains blue-black, while waxy starch stains red-brown.
  • waxy wheat high amylopectin wheat
  • glues which may not require as high amounts of emulsifiers and would likely have improved consumer reception.
  • livestock industry is interested in obtaining waxy varieties of cereals that are used as livestock feed because the greater branching structure of amylopectins makes the molecules more readily degradable by amylolytic enzymes, thus increasing the rate of energy availability to monogastric animals.
  • Suitable target genes include the genes that are involved in the biosynthesis of phytic acid. All seeds of both legumes and cereals contain phytic acid as the main storage form of phosphate in the seeds.
  • Phytic acid is a myoinositol hexakisphosphate, which is important in seed germination and early development, but appears not to be essential to the plant, since inorganic phosphate can be accumulated to serve essentially the same function (Raboy, V., The Biochemistry and Genetics of Phytic Acid Synthesis. 1990. pp. 52-73. In: Inositol Metabolism in Plants. Morre, D.J., Boss W.F. and Loewus, F. A. (eds).
  • Phytic acid is also a very strong chelator of divalent mineral ions, such as Zn, Cu, Ca, Fe and Co, and is responsible for the excretion of the seed phosphates and minerals by animals, i.e., the phosphates and minerals are bound to the phytic acid and so are inaccessible for absorption by the gut.
  • the animal excretions of phytic phosphate chelated minerals results in about 30%> of the manure produced by poultry, and perhaps a similar amount produced by swine. These excess excrements not only contribute to environmental pollution, but also deny the animals of the mineral nutrients, which must be supplied from other sources. Null mutations of the genes controlling the enzymes responsible for phytic acid synthesis in seeds would prevent loss of the minerals from animal diets, reduce the amount of mineral pollutants released into the environment from manure and improve the utilization of minerals and phosphates from seeds fed to livestock.
  • Suitable target genes include genes encoding oxidases, upases and lipoxygenases.
  • oxidases oxidases
  • upases oxidases
  • lipoxygenases oxidases
  • lipoxygenases oxidases, upases and lipoxygenases.
  • oat endosperm contains on average 5-6% oil, and the natural variation in the levels of oat seed oil suggests that the oil level could be increased to 16-18%, making oats a commercial oil source, if rancidity of the oil could be prevented by inactivating the degradative enzymes.
  • oat oil contains a high proportion of tocopherol which is an important source of vitamin E.
  • Tocopherol levels could also be enhanced by eliminating or reducing endogenous lipase and/or lipoxygenase activity.
  • a convenient and simple screening system for mutants having reduced or no lipoxygenase/lipase activity could be based on the ability of lipoxygenases/lipases to oxidize carotenoids (Hildebrand, D.E. et al. Current Topics in Plant Biochemistry and Physiology 7:201-219 (1988)).
  • lipoxygenase and lipase are not essential to the growth of the plants, though these enzymes may function in insect or disease resistances (Hildebrand, et al, J. Econ. Entom. 79: 1459-1465 (1986)). It may be possible to delete these enzymes from the seeds, without affecting their activity in the plants. These enzymes apparently affect the flavor of products made from soybeans. However, natural soybean mutants were found in the soybean germplasm, permitting the breeding of soybean cultivars lacking the peroxidases (Hildebrand, D.E. et al. Current Topics in Plant Biochemistry and Physiology 7:201-219 (1988)).
  • durum wheats these enzymes cause the breakdown of carotenoids, which are highly important for pasta quality (Matsuo et al, Cereal Chem. 47:1(1970); Lee, et al., Theor. Appl. Genet. 47:243 (1976); McDonald, C.E., Cereal Chem. 56:84 (1979); Laignelet, B., Sci. Aliment. 3:469 (1983).
  • Durums have been developed for increased carotenoid content of the flour or semolina from the endosperm, and over time cultivars have been developed with comparatively high levels of carotenoids. Such high levels of carotenoids have been achievable only by reducing the destruction of the pigment by lipoxygenases.
  • these enzymes are not essential to the growth and vigor of the plants, but may still exist in crop species as relics from evolution for which they served to assure continued survival of the species by making the seed less palatable to wild animals.
  • the chromosomal location of peroxidases in durum wheats is known, and tests for the activity of the enzymes are available (Kobrehel, K. and Fiellet, P., Can. J. Bot. 53:2336 (1975); Hseih, C.C. and McDonald, C.E., Cereal Chem. 61:392 (1984)).
  • a sample of crude semolina With durums, exposure of a sample of crude semolina to moist air for a period of hours is enough to cause loss of yellow pigment color, if peroxidase activity is present.
  • Carotenoids are too oxygen-sensitive for use in a screening method, but some other luteins may prove useful as detectors of peroxidase activity in oats. Reports in the literature indicate that methyl-jasmonate may be useful to accelerate the reaction.
  • Another example of a suitable target gene isoform family includes polyphenol oxidases. Polyphenol oxidases occur almost universally in plants. These oxidases are responsible for the browning reaction of cut surfaces and bruises in apples and peaches.
  • durum breeders have discovered natural genetic variability for null alleles of the polyphenol oxidases in durums
  • no genetic source of null polyphenol oxidase genes is available, though several wheat genetic stocks have been identified that have low polyphenol oxidase levels (Bernier, A.M. and Howes, N.K., J. Cereal Sci. 19: 157-159 (1994)).
  • Canadian scientists have discovered a null locus for the enzyme in the diploid D-genome progenitor of hexaploid wheat, T.
  • leyii hence have synthesized a genetic source of zero polyphenol oxidase in hexaploid wheat (Bernier and Howes, 1997, personal communication).
  • the wheat they developed is a synthetic, carrying many genes from the wild progenitor and the durum parent, the synthetic is not as useful as a source of the null alleles as would be a mutant induced in one of the modern hexaploid wheats with a low polyphenol oxidase activity.
  • a simple and rapid screening system has been developed (Bernier, A.M. and Howes, N.K., J. Cereal Sci.
  • Wheat-Triticum turgidum durum tetraploid cultivated forms, which include durum (pasta wheats) and a special large kernel form, Triticum turgidum turanicum.
  • Triticale- Triticale hexaploide Wittmack. Hexaploid triticale a modern synthetic species constructed from T. turgidum (durum) x Secale cereale L. (rye). Cotion-Gossypium hirsutum L. now known to be a natural synthetic tetraploid constructed of G. arboreum L. x G. barbadense L.
  • MMfa-Medicago sativa L. is an allotetraploid (like cotton) constructed from M. falcata L. x M. glutinosa L-, which often shows tetrasomic inheritance of traits, indicative of genetic homeology Tob&cco-Nicotiana tabacum L. an allotetraploid of natural origin, combining genomes from N. sylvestris Speg. x N. tomentosiformis Goodsp. The genomes are similar enough to often show tetrasomic inheritance of some traits. hypogea L. is a probable allotetraploid for which the diploid progenitors are uncertain. However, the species carries traits, which indicate homeology between genomes (sets) of 10 chromosome pairs.
  • Coffee-Caf ea arabica L. and Caffea canephora L are both tetraploid species, which like most others mentioned carry sets of chromosomes with much homeology
  • Rapeseed and MxsX& ⁇ s-Brassica napus. L ssp. oleifera (Metzg.) (Summer and winter rapeseed.), and Brassica juncea L. (brown, oriental mustard) are allotetraploids. Many grass species are polyploids, as are a number of other legumes, including Birdsfoot trefoil, Lotus corniculatus L.;
  • Cicer milkvetch Astragalus cicer L.; and Lupines, Lupinus albus L.,
  • EXAMPLE 1 Generation of Waxy Mutants of Wheat Line Kanto 107 The seeds (about 2.5 kg) of Kanto 107 wheat (carrying null waxy mutants in the A and B genomes) were presoaked (immersed in water) for 4-5 hr. prior to the mutagen treatments. The water used was tap water (although distilled water may be used.). After the presoaking period, the water was poured off, and a liter of distilled water was added to each container of seeds. The mutagen ethyl methane sulfonate (EMS) was applied to the seeds in the distilled water solution.
  • EMS mutagen ethyl methane sulfonate
  • the treatments applied included one treatment at 0.2 milliliter EMS per liter of distilled water, and two treatments at 0.25 milliliter of mutagen (EMS) per liter of distilled water.
  • the seeds were allowed to soak in each of the mutagen solutions for 2 hours and 15 min., with shaking of the treatment containers every 15-20 min. during treatment to improve the contact of the seeds with the mutagen solutions.
  • This azide treatment was continued with intermittent shaking (every 15 min.) for 1 hour, after which the solution was poured off into the disposal container, and the seeds in each container were given a distilled water rinse. After rinsing, the seeds were placed to redry in screen baskets in a fume hood. Redrying in the fume hood was continued for 14 hours, then the seeds were taken to another laboratory to continue redrying at room temperature for another 24 hours. At this point, the treated seeds were placed in seeder magazines and planted in the field, much as any seeds would be sown for production.
  • seed sample 0 is the original Kanto 107 variety which carries two waxy null gene loci; seed samples 1 and 4-25 are waxy mutants derived from mutagenized Kanto 107 seed; seed sample 3 is a partially- penetrant waxy mutant, i.e., a mutant which does not show the full waxy phenotype; seed sample 26 is a soft white, spring wheat, cv Penawawa, which carries a single waxy null gene at the B locus, and seed sample 27 is a soft white, spring wheat, cv
  • Prime starch is the essentially pure starch fraction, containing only unbroken starch granules, and separated from the principal protein fractions in the flour, it has a very low ash content and is very low in protein
  • the prime starch still contains small amounts of phospholipids and a very minor fraction of proteins mostly associated with the starch granules.
  • the data include the temperature at which the peak (highest) viscosity occurs, the actual recorded viscosity of the solubilized starch at the peak temperature, the viscosity 5 minutes after the peak viscosity was reached, the viscosity at the end of a period of 30 minutes holding at the peak temperature, and at the end of a period of 30 minutes holding the gelled starch at 50°C
  • Kanto 107 mutant 3 appears to be a partially waxy line, but it was first identified as waxy, like all the rest, but the seed harvested from the M2 and M3 plants proved not to stain red like typical waxy mutants, thus was thought to have been selected in error, but seed of it was increased anyway in order to have a single line selection from Kanto 107.
  • mutant 3 has many properties similar or intermediate between those of Kanto 107 (0), and the truly waxy mutants 1, 4-25.
  • Mutant 3 must contain a fair amount of amylose, since its peak gelatinization temperature is much like that of Penawawa, though its peak viscosity is more nearly like that of Kanto 107.
  • amylopectin content is presumably intermediate between that of Kanto 107 and a more fully waxy mutant, having nearly 99%o amylopectin.
  • peak viscosity data for mutants 9 and 21.
  • the gels of these mutants have a rather high viscosity at their peak temperature of gelling.
  • Table 2 the texture of gels made from the prime starch of the mutants and controls was measured after storing the gels at 4°C for 96 hr. after gelling.
  • mutants 9 and 21 started at modest levels, and changed very little over time. Some other mutants followed a similar pattern, but began at either lower or higher texture levels.
  • the stability over time and temperature of gels made from starches is an extremely important factor in the selection of materials for food products.
  • mutant 3 shows an unusual gel texture under the test conditions. Although the gel made from its prime starch had an initial texture slightly higher than that of Kanto 107, the gel texture increase much less over time of storage.
  • the starches from these mutants are not all alike, thus they offer the potential for developing unique wheat starch sources for special industrtial or food products.
  • mutants also appear to have distinctly different polymer compositions based on analyses by High Performance Size Exclusion Chromatograpy, after alpha amylase debranching of the starch polymers.
  • FIGURES 1-3 the sizes of the polymers in the starches from some of the mutants listed in Tables 1 and 2, in relation to their degree of polymerization, differ notably.
  • FIGURE 1 shows that mutant 21 has a greater proportion of higher molecular weight polymers than does mutant 18.
  • mutant 22 has more low molecular weight starch polymers
  • mutant 4 has a distinctly higher proportion of higher molecular weight polymers in its make-up.
  • mutants have been induced, since all mutants described are expected to be at the same D-genome waxy locus.
  • electrophoretic analyses of the waxy Kanto 107 mutants show that some of the mutants, of those so far analysed, have an apparently non- or less- functional D-genome GBSS protein, while some others, though they produce a different spectrum of high molecular weight amylopectin polymers, appear to be null locus mutants.
  • mutants 5, 7, 8, 11, 12, 14, 15, and 18 appear to have no protein at the position typical for the D-GBSS protein band
  • Mutants 1, 9, 13, 16, and 17 appear to have a fairly strong D-genome waxy protein band.
  • Mutants 4, 6, and 10 have a faint protein band at the D-genome Wx protein band position. More detailed analyses may yet show that some of the apparently null mutants carry proteins, which move to a different position in the electrophoresis gels. Nevertheless, it is quite clear that the different mutants result from a variety of alteration in the gene locus controlling the production of the D-genome GBSS protein. Those mutants with D-genome protein bands clearly must carry modifications in the GBSS enzyme which make them differentially functional or non-functional in their debranching capability. Similarly those mutants with a faint level of the protein must also be able to produce enough of a non- or nearly- nonfunctional enzyme to show up in the electrophoresis gels.

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Abstract

L'invention concerne des procédés qui permettent de générer et d'identifier des mutations dans n'importe quel gène cible d'une espèce de plante polypoïde. Dans un aspect préféré de l'invention, on décrit une plante construite et/ou choisie possédant au moins une copie d'un gène cible fonctionnel situé exclusivement sur un seul de ses génomes homéologues ou homologues. Des semences provenant de la plante choisie sont ensuite placées au contact d'une quantité effective d'au moins un agent mutagène. Les semences traitées sont germées et les semences ou plantes ainsi produites sont criblées pour identifier des mutations dans le gène cible. Ainsi, les concepts définis dans l'invention peuvent être mis en oeuvre pour créer, sélectionner et identifier des mutations dans n'importe quel gène cible d'une quelconque plante polypoïde appropriée, ce qui permet d'obtenir une source de mutations nombreuses, facilement identifiables pouvant être utilisées, au besoin, dans des croisements destinés à la mise au point des cultivars de plante nouveaux et uniques. De nouveaux allèles de gènes cibles homéologues ou homologues peuvent être recombinés pour générer une gamme presqu'illimitée de nouveaux phénotypes génétiquement déterminés présentant des propriétés désirables préétablies.
PCT/US1999/013801 1998-06-19 1999-06-18 Procedes pour generer et identifier des plantes polypoides mutantes et utilisation desdites plantes WO1999065292A1 (fr)

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EP1708559A2 (fr) * 2003-12-03 2006-10-11 Arcadia Biosciences, Inc. Ble comprenant une proteine cireuse reduite du fait d'alterations non transgeniques d'un gene cireux
WO2005072186A3 (fr) * 2004-01-21 2007-02-01 Omega Genetics Llc Plantes tolerant le glyphosate et procedes de fabrication et d'utilisation
WO2009041810A1 (fr) * 2007-09-24 2009-04-02 Keygene N.V. Procédé de sélection de végétaux comportant des mutations spécifiques
US20120266333A1 (en) * 2001-08-09 2012-10-18 Pierre Hucl Wheat plants having increased resistance to imidazolinone herbicides
US8389809B2 (en) * 2001-08-09 2013-03-05 Basf Se Wheat plants having increased resistance to imidazolinone herbicides
WO2014110274A3 (fr) * 2013-01-09 2015-02-26 Regents Of The University Of California A California Corporation Génération de plantes haploïdes
US9035133B2 (en) 2006-12-12 2015-05-19 Basf Agrochemical Products B.V. Herbicide-resistant sunflower plants and methods of use
US10017827B2 (en) 2007-04-04 2018-07-10 Nidera S.A. Herbicide-resistant sunflower plants with multiple herbicide resistant alleles of AHASL1 and methods of use
CN109089877A (zh) * 2018-07-10 2018-12-28 山西省农业科学院玉米研究所 一种利用ems诱变产生芸豆或小扁豆突变体的方法
CN109729972A (zh) * 2019-01-15 2019-05-10 山西省农业科学院小麦研究所 一种提高小麦ems诱变率的方法
CN111996177A (zh) * 2020-08-17 2020-11-27 北京市农林科学院 一种玉米waxy基因突变体及其分子标记和应用
CN115968777A (zh) * 2023-02-13 2023-04-18 广西壮族自治区农业科学院 一种玉米同源四倍体的制作方法

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Cited By (21)

* Cited by examiner, † Cited by third party
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US11746343B2 (en) 2001-08-09 2023-09-05 Northwest Plant Breeding Company Wheat plants having increased resistance to imidazolinone herbicides
US20120266333A1 (en) * 2001-08-09 2012-10-18 Pierre Hucl Wheat plants having increased resistance to imidazolinone herbicides
US8389809B2 (en) * 2001-08-09 2013-03-05 Basf Se Wheat plants having increased resistance to imidazolinone herbicides
US20130190180A1 (en) * 2001-08-09 2013-07-25 Northwest Plant Breeding Company Wheat plants having increased resistance to imidazolinone herbicides
US9879235B2 (en) * 2001-08-09 2018-01-30 University Of Saskatchewan Wheat plants having increased resistance to imidazolinone herbicides
US9499833B2 (en) * 2001-08-09 2016-11-22 Northwest Plant Breeding Company Wheat plants having increased resistance to imidazolinone herbicides
EP1708559A2 (fr) * 2003-12-03 2006-10-11 Arcadia Biosciences, Inc. Ble comprenant une proteine cireuse reduite du fait d'alterations non transgeniques d'un gene cireux
EP1708559A4 (fr) * 2003-12-03 2010-01-06 Arcadia Biosciences Inc Ble comprenant une proteine cireuse reduite du fait d'alterations non transgeniques d'un gene cireux
US8735649B2 (en) 2003-12-03 2014-05-27 Arcadia Biosciences, Inc. Wheat having reduced waxy protein due to non-transgenic alterations of a waxy gene
WO2005072186A3 (fr) * 2004-01-21 2007-02-01 Omega Genetics Llc Plantes tolerant le glyphosate et procedes de fabrication et d'utilisation
US9035133B2 (en) 2006-12-12 2015-05-19 Basf Agrochemical Products B.V. Herbicide-resistant sunflower plants and methods of use
US10017827B2 (en) 2007-04-04 2018-07-10 Nidera S.A. Herbicide-resistant sunflower plants with multiple herbicide resistant alleles of AHASL1 and methods of use
US8716550B2 (en) 2007-09-24 2014-05-06 Keygene N.V. Method for the selection of plants with specific mutations
WO2009041810A1 (fr) * 2007-09-24 2009-04-02 Keygene N.V. Procédé de sélection de végétaux comportant des mutations spécifiques
WO2014110274A3 (fr) * 2013-01-09 2015-02-26 Regents Of The University Of California A California Corporation Génération de plantes haploïdes
CN109089877A (zh) * 2018-07-10 2018-12-28 山西省农业科学院玉米研究所 一种利用ems诱变产生芸豆或小扁豆突变体的方法
CN109089877B (zh) * 2018-07-10 2021-11-09 山西省农业科学院玉米研究所 一种利用ems诱变产生芸豆或小扁豆突变体的方法
CN109729972A (zh) * 2019-01-15 2019-05-10 山西省农业科学院小麦研究所 一种提高小麦ems诱变率的方法
CN111996177A (zh) * 2020-08-17 2020-11-27 北京市农林科学院 一种玉米waxy基因突变体及其分子标记和应用
CN111996177B (zh) * 2020-08-17 2022-03-04 北京市农林科学院 一种玉米waxy基因突变体及其分子标记和应用
CN115968777A (zh) * 2023-02-13 2023-04-18 广西壮族自治区农业科学院 一种玉米同源四倍体的制作方法

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