WO2012122019A1 - Barley cultivar moravian 115 - Google Patents

Barley cultivar moravian 115 Download PDF

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Publication number
WO2012122019A1
WO2012122019A1 PCT/US2012/027479 US2012027479W WO2012122019A1 WO 2012122019 A1 WO2012122019 A1 WO 2012122019A1 US 2012027479 W US2012027479 W US 2012027479W WO 2012122019 A1 WO2012122019 A1 WO 2012122019A1
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barley
plant
plants
gene
resistance
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PCT/US2012/027479
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French (fr)
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Dennis DOLAN
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Millercoors, Llc
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Publication of WO2012122019A1 publication Critical patent/WO2012122019A1/en

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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10Seeds
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4624Hordeum vulgarus [barley]

Definitions

  • the present invention relates to a new and distinctive barley cultivar designated Moravian 115 (MVl 15). All publications cited in this application are herein incorporated by reference.
  • Barley (Hordeum vulgare L.) is a grain that is grown worldwide with three main market classes, malt, feed and food. Most of the barley grain produced in the United States is used as an ingredient in cattle, pig, or poultry feed. Another major use for barley is malt production. Malt is used in the brewing and distilling industries to produce alcoholic beverages. Barley varieties that are preferred for producing malt are selected on the basis of characteristics such as kernel plumpness, low protein content and low Beta-glucan content. Barley grain that has more than about 13.5 weight percent protein on a dry basis or is too dark in color is rejected by malting plants. Significant overlap between the classes can occur since barley that does not meet malting specifications can be used for feed, food and potentially the emerging biofuels industry.
  • Barley is a nutritious food ingredient for humans or household pets. When used as a food ingredient, malting or feed barley grain that has a cemented hull (referred to as covered) must be processed to remove that hull. A commonly used processing step known as pearling removes the hull and a substantial portion of the bran and the germ to produce a pearled barley grain, such that at least about 15 to about 40 weight percent of the outer grain is removed. Barley varieties developed especially for food are hulless, i.e., they have a loose hull so do not have to be pearled prior to consumption. Hulless barley must be cleaned as do all grains prior to entering the human food markets, but loose hulls can be removed easily with only slight modifications to the cleaning plants. Food ingredient manufacturers may grind the cleaned barley to produce flour or roll the barley to produce flakes. Food ingredient manufacturers may also utilize the cleaned barley as a whole berry (seed).
  • Waxy barley is a naturally occurring variant that has recently been investigated for potential in food and industrial processing. Barley lines having the waxy phenotype have reduced amounts of amylose starch in the seed. The waxy trait may be useful in the production of high maltose syrup from barley (U.S. Pat. No. 4,116,770, Goering 1978) and in the production of flour and flakes (U.S. Pat. No. 5,614,242, Fox 1997 and U.S. Pat. No. 6,238,719, Fox, 2001) that have health benefits. [0005] The health promoting benefits of barley consumption have been investigated in human clinical trials.
  • Cultivated barley is a naturally self-fertilizing species, although there is a small percentage of cross-fertilization.
  • Natural genetic and cytoplasmic male sterility is available to use in breeding and in hybrid seed production. Using all of the tools available to a breeder, it is possible to develop pure lines that are uniform in growth habit, maturity, yield, and other qualitative and quantitative characteristics. These lines can be released as inbred varieties, as inbreds for hybrid barley, or as lines to be further manipulated in the development of new lines or varieties or that incorporate proprietary genetic material.
  • Barley varieties may differ from each other in one or more traits and can be classified and differentiated according to the specific traits they possess. For example, there are types of barley known as two-rowed and other types known as six-rowed, referring to the number and positioning of kernels on the spike. Barley lines also can be classified as spring barley or winter barley, referring to the growth habit, or by the adherence of hulls on the seed, or by the type of starch in the seed. There are, of course, many other traits, which differentiate the various lines. A discussion of breeding methods for developing barley lines and of some traits in barley can be found in Foster, A. E., Barley, pp. 83-125, and in Fehr, W.
  • cultivars are not necessarily pure lines (they could be a mixture of genotypes or even be a hybrid) presently, most named barley cultivars are pure lines.
  • the present invention relates to a new and distinctive barley variety, designated Moravian 115 (MV115), which has been the result of years of careful breeding and selection as part of a barley breeding program.
  • MV115 Moravian 115
  • Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives.
  • the next step is selection of germplasm that possess the traits to meet the program goals.
  • the goal is to combine in a single variety an improved combination of desirable traits from the parental germplasm.
  • These important traits may include higher seed yield, resistance to diseases and insects, tolerance to drought and heat, better agronomic qualities and improved grain quality.
  • Field crops are bred through techniques that take advantage of the plant's method of pollination.
  • a plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant.
  • a plant is sib-pollinated when individuals within the same family or line are used for pollination.
  • a plant is cross-pollinated if the pollen comes from a flower on a different plant from a different family or line.
  • the term cross- pollination herein does not include self-pollination or sib-pollination.
  • a cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci.
  • a cross of two heterozygous plants each that differ at a number of gene loci will produce a population of plants that differ genetically and will not be uniform. Regardless of parentage, plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny.
  • the term "homozygous plant” is hereby defined as a plant with homozygous genes at 95% or more of its loci.
  • breeding term "inbred” as used herein refers to a homozygous plant or a collection of homozygous plants.
  • Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of variety used commercially (e.g., Fi hybrid variety, pureline variety, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants.
  • Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.
  • Pedigree breeding is commonly used for the improvement of self-pollinating crops. Two parents that possess favorable, complementary traits are crossed to produce an Fi . An F2 population is produced by selfing or sibbing one or several FiS. Selection of the best individuals may begin in the F 2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families can begin in the F 4
  • Each barley breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful varieties produced per unit of input (e.g., per year, per dollar expended, etc.).
  • recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes.
  • the use of recurrent selection in self-pollinating crops depends on the ease of pollination and the number of hybrid offspring from each successful cross.
  • Recurrent selection can be used to improve populations of either self- or cross-pollinated crops.
  • a genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued. Plants from the populations can be selected and selfed to create new varieties.
  • Another breeding method is single-seed descent.
  • This procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation.
  • the plants from which lines are derived will each trace to different F 2 individuals.
  • the number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed.
  • not all of the F 2 plants originally sampled in the population will be represented by a progeny when generation advance has been completed.
  • barley breeders commonly harvest one or more spikes
  • the procedure has been referred to as modified single-seed descent.
  • the multiple-seed procedure has been used to save labor at harvest. It is considerably faster to thresh spikes with a machine than to remove one seed from each by hand for the single-seed procedure.
  • the multiple-seed procedure also makes it possible to plant the same number of seeds of a population each generation of inbreeding. Enough seeds are harvested to make up for those plants that did not germinate or produce seed.
  • Bulk breeding can also be used.
  • an F 2 population is grown.
  • the seed from the populations is harvested in bulk and a sample of the seed is used to make a planting the next season. This cycle can be repeated several times.
  • individual plants are expected to have a high degree of homozygosity, individual plants are selected, tested, and increased for possible use as a variety.
  • Molecular markers including techniques such as Starch Gel Electrophoresis, Isozyme Eletrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple
  • SSRs Sequence Repeats
  • SNPs Single Nucleotide Polymorphisms
  • QTL mapping is the use of markers, which are known to be closely linked to alleles that have measurable effects on a quantitative trait. Selection in the breeding process is based upon the accumulation of markers linked to the positive effecting alleles and/or the elimination of the markers linked to the negative effecting alleles from the plant's genome.
  • markers can also be used during the breeding process for the selection of qualitative traits. For example, markers closely linked to alleles or markers containing sequences within the actual alleles of interest can be used to select plants that contain the alleles of interest during a backcrossing breeding program. The markers can also be used to select for the genome of the recurrent parent and against the markers of the donor parent. Using this procedure can minimize the amount of genome from the donor parent that remains in the selected plants. It can also be used to reduce the number of crosses back to the recurrent parent needed in a backcrossing program (Openshaw et al. Marker- assisted Selection in Backcross Breeding. In: Proceedings Symposium of the Analysis of Molecular Marker Data, 5-6 Aug. 1994, pp. 41-43. Crop Science Society of America, Corvallis, Oreg.). The use of molecular markers in the selection process is often called Genetic Marker Enhanced Selection.
  • Double haploids are produced by the doubling of a set of chromosomes (IN) from . a heterozygous plant to produce a completely homozygous individual. This can be advantageous because the process omits the generations of selfing needed to obtain a homozygous plant from a heterozygous source.
  • Various methodologies of making double haploid plants in barley have been developed (Laurie, D. A. and S. Reymondie, Plant Breeding, 1991, v. 106:182-189. Singh, N. et al, Cereal Research Communications, 2001, v. 29:289-296; Redha, A. et al., Plant Cell Tissue and Organ Culture, 2000, v. 63:167-172; U.S. Pat. No. 6,362,393)
  • hybrid barleys are produced with the help of cytoplasmic male sterility, nuclear genetic male sterility, or chemicals. Various combinations of these three male sterility systems have been used in the production of hybrid barley.
  • a breeder uses various methods to help determine which plants should be selected from the segregating populations and ultimately which lines will be used for
  • Plant breeding is the genetic manipulation of plants.
  • the goal of barley breeding is to develop new, unique and superior barley varieties.
  • the breeder initially selects and crosses two or more parental lines, followed by repeated selfing and selection, producing many new genetic combinations.
  • the breeder can theoretically generate billions of different genetic combinations via crossing, selection, selfing and mutations. Therefore, a breeder will never develop the same line, or even very similar lines, having the same barley traits from the exact same parents.
  • Barley is an important and valuable field crop.
  • a continuing goal of barley breeders is to develop stable, high yielding barley varieties that are agronomically sound and have good grain quality for its intended use.
  • the barley breeder must select and develop barley plants that have the traits that result in superior varieties.
  • a new barley cultivar designated Moravian 115 (MVl 15).
  • This invention thus relates to the seeds of barley cultivar MVl 15, to the plants of barley cultivar MVl 15 and to methods for producing a barley plant produced by crossing the barley cultivar MV115 with itself or another barley cultivar, and the creation of variants by mutagenesis or transformation of barley cultivar MVl 15.
  • any such methods using the barley cultivar MVl 15 are part of this invention: selling, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using barley cultivar MVl 15 as at least one parent are within the scope of this invention.
  • the barley cultivar could be used in crosses with other, different, barley plants to produce first generation (Fi) barley hybrid seeds and plants with superior characteristics.
  • the present invention provides for single or multiple gene converted plants of barley cultivar MVl 15.
  • the transferred gene(s) may preferably be a dominant or recessive allele.
  • the transferred gene(s) will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, male sterility, enhanced nutritional quality, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified protein percent, modified beta-glucan percent, modified lodging resistance, modified lipoxygenase, beta- glucanase and/or polyphenol oxidase content and/or activity, and industrial usage.
  • the gene may be a naturally occurring barley gene or a transgene introduced through genetic engineering techniques.
  • the present invention provides regenerable cells for use in tissue culture of barley plant MVl 15.
  • the tissue culture will preferably be capable of regenerating plants having essentially all the physiological and morphological characteristics of the foregoing barley plant, and of regenerating plants having
  • the regenerable cells in such tissue cultures will be selected or produced from head, awn, leaf, pollen, embryo, cotyledon, hypocotyl, seed, spike, pericarp, meristematic cell, protoplast, root, root tip, pistil, anther, floret, shoot, stem and callus.
  • the present invention provides barley plants regenerated from the tissue cultures of the invention.
  • Alpha-amylase activity of malt per dry weight of malt is an endogenous enzyme of barley that is produced during germination. It degrades malt starch to dextrins. It is also called liquefying enzyme as it reduces viscosity of gelatinized starch solution.
  • Awn is intended to mean the elongated needle-like appendages on the flower-and seed-bearing "head" at the top of the barley plant. These awns are attached to the lemmas. Lemmas enclose the stamen and the stigma as part of the florets. These florets are grouped in spikelets, which in turn together comprise the head or spike.
  • Beta-glucan Dry Basis beta-glucan is barley cell wall component that is degraded during germination to enable starch extraction from the grain.
  • the beta-glucan content of malt indicates the completeness of cell wall modification. The lower the beta-glucan content, the more complete cell wall degradation is presumed. High beta-glucan content of malt can impede lautering and beer filtration.
  • Beta-glucan fiber is a nonstarch polysaccharide in which individual glucose molecules (20,000-1,000,000) are linked by beta 1-4 and beta 1-3 linkages. Beta-glucan is soluble in warm water (40-45 degrees Centigrade); cellulose is insoluble in water. Beta-glucan is the main structural material in the cell walls of barley and oat grain.
  • Beta-glucan fiber viscosity describes the friction that is created in a solution by the presence of beta-glucan chains (fibers) and is measured in centipoise units.
  • Centipoise units are the units commonly used to measure viscosity.
  • the fundamental unit of viscosity measurement is the "Poise”, which is a material requiring a sheer stress of one dyne per square centimeter to produce a sheer of one inverse second, which has a viscosity of one poise or 100 centipoise.
  • Covered seed Barley seed can have a cutin layer which cements the hull (lemma and palea or glumes) to the seed. This trait is controlled by the Nud locus on chromosome 1 (7H). The homozygous dominant Nud Nud genotype results in the presence of cutin and is referred to as covered. The hull can only be removed by abrasive processing prior to consumption, known as pearling.
  • DP Dry Basis Diastatic Power (DP) value indicates the malt enzyme activity that degrades dextrins and starch to fermentable sugars.
  • Extract value indicates the percentage of dry malt matter that can be solubilized to water during mashing. It is determined by measuring specific gravity of wort.
  • a plant having essentially all of the physiological and morphological characteristics means a plant having the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted trait.
  • FAN Dry Basis Free Amino Nitrogen (FAN) consists of free amino acids and small peptides. They are produced from barley proteins by proteolytic eruzymes during malting and mashing.
  • Foliar disease is a general term for fungal disease which causes yellowing or browning or premature drying of the leaves. The disease typically involves Septoria, net blotch, spot blotch or scald.
  • Gene refers to a segment of nucleic acid.
  • a gene can be introduced into a genome of a species, whether from a different species or from the same species, using transformation or various breeding methods.
  • head refers to a group of spikelets at the top of one plant stem.
  • spike also refers to the head of a barley plant located at the top of one plant stem.
  • Lodging refers to the bending or breakage of the plant stem, or the tilting over of the plant, which complicates harvest and can diminish the value of the harvested product.
  • Leaf rust A fungal disease that results in orange-red pustules on the leaf surface. Caused by Puccinia hordei.
  • Malt Protein Total protein content of malt. Typically 40-50% of the malt protein is soluble in wort.
  • Maturity Maturity, higher is better (FEB) refers to the physiological stage of plant growth used to describe early or lateness of the variety. Maturity is measured on a scale of 3- 1. The higher the maturity number the earlier maturing the variety.
  • Net blotch refers to a fungal disease which appears as elongated black lesions running parallel to the leaf veins with distinctive, dark brown net-like patterns. Net blotch is caused by Pyrenophora teres.
  • Plant includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. A seed or embryo that will produce the plant is also considered to be the plant.
  • Plant height As used herein, the term “plant height” is defined as the average height in inches or centimeters of a group of plants, as measured from the ground level to the tip of the head, excluding awns.
  • Plant parts As used herein, the term “plant parts” (or a barley plant, or a part thereof) includes but is not limited to protoplasts, leaves, stems, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, flower, shoot, tissue, petiole, cells, meristematic cells, head, awn, spike, pericarp, pistil, and callus and the like.
  • Plump (%) Refers to the percentage of grain retained on a 6/64" screen when shaken on a timed mechanical shaker for 15 revolutions. This test measures the fraction of kernels that are termed "plump grain”.
  • progeny includes an barley plant produced from the cross of two barley plants where at least one plant includes barley cultivar Moravian 115 and progeny further includes but is not limited to subsequent F 2 , F 3 , F 4 , F5, F6, F 7 , Fg, F 9 and F10 generational crosses with the recurrent parental line.
  • Progeny also includes Si plant produced from the selfing of barley cultivar Moravian 115; progeny further includes but is not limited to subsequent selfing generations and crosses with the recurrent parental line.
  • Regeneration refers to the development of a plant from tissue culture.
  • Scald refers to a fungal disease that causes spots to develop on the leaves during cool, wet weather. The spots are oval shaped and the margins of the spots change from bluish-green to zonated brown or tan rings with bleached straw-colored centers. Scald is caused by Rkynchosporium secalis.
  • Septoria Septoria refers to a fungal disease that appears as elongated, light brown spots on the leaves. It is caused by Septoria passerinii.
  • Single gene converted (Conversion) plants refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique, genetic engineering or mutation, either induced or spontaneous.
  • Covered smut refers to a fungal disease in which masses of black spores replace the seed kernels on the head. A persistent membrane can be ruptured during harvest to disperse spores. Covered smut is caused by Ustilago hordei.
  • Loose smut refers to a fungal disease in which masses of black spores replace the seed kernels on the head. The thin membrane that covers the spores is easily ruptured and spores disbursed by wind. Loose smut is caused by Ustilago nuda.
  • Spot blotch Spot blotch refers to a fungal disease that appears as dark, chocolate- colored blotches forming irregular dead patches on the leaves. Spot blotch is caused by Cochliobolus sativus.
  • Stem rust refers to a fungal disease that produces masses of brick-red pustules on stems and leaf sheaths. Stem rust can be caused by either Puccinia graminis f. sp. tritici or Puccinia graminis f. sp. secalis.
  • Stripe rust refers to a fungal disease that results in light yellowish orange pustules arranged in stripes between the veins of the leaves. Stripe rust is caused by Puccinia striiformis f. sp. hordei.
  • Test Weight The test weight per bushel (U.S.) is the weight (in pounds) of the volume of grain required to fill level full a Winchester bushel measure of 2,150.42 cubic inches capacity" ( ef. 2, Chap. XI). This translates into pounds per 32 U.S. dry quarts or pounds per 35.238 L.
  • Viscosity Resistance of fluid to flow. Wort viscosity indicates the presence of soluble dextrins, cell wall beta-glucans or gums in the wort that can impede lautering and beer filtration.
  • Moravian 115 (previously also known as CI 15) is a small-statured, high yielding, two-rowed malting barley with high extract, moderate protein (10.5-12.5%) and bright, plump seed that originated from the cross C97-73-05/C57. Both parents are high yielding malting lines.
  • the Fi was grown near Burley, Idaho in the winter of 2001 and 2002 and the F 2 and F3 plants were greenhouse grown near Burley, Idaho in the spring and fall, respectively of 2002.
  • Single F 4 plants were selected in the summer of 2003 and one such plant was given the designation C01-47-14. This single plant was planted as an individual single row in a winter nursery near Leeston, New Zealand in 2003-2004.
  • F4:F6 single, un-replicated plots were produced near Burley, Idaho in the spring and summer of 2004. Seed from this single plot was used for malting and for producing replicated plots in 2005. During subsequent increases and evaluations through the F6 to Fn generations, Moravian 115 proved to be stable and uniform.
  • Moravian 115 is a semi-dwarf, two-rowed, spring habit malting barley variety selected from a breeding population for high yield and exceptional malting characteristics. Moravian 115 will be used as malting barley for the brewing industry.
  • Moravian 115 is a two-rowed, medium-maturing, short variety that has an early prostrate growth habit and has medium length awns that is adapted to the high desert areas of the Mountain States. Moravian 115 was selected for high agronomic performance and malting quality. The agronomic and malting characteristics of Moravian 115 are listed in Table 1. Comparisons between Moravian 115 and other Moravian barley lines are in Tables 2-5.
  • the spike of Moravian 115 is two-rowed, has a straight neck, a closed collar, is non-waxy, and erect at maturity.
  • the spike has a few hairs on the rachis edge.
  • the glumes of Moravian 115 are less than equal to the length of the glume, have a band of short hairs, and are awnless.
  • the lemma has medium length awns that are rough.
  • the base of the lemma has a depression and the rachilla has many long hairs. Seeds of Moravian 115 are hulled with a white aleurone, are midlong and have wrinkled, loosely attached hulls.
  • the spike of Moravian 115 typically does not emerge completely from the flag leaf sheath or boot. Typically, Moravian 115 is most successfully produced under irrigated conditions and does not lodge under normal production conditions.
  • Maturity (50% flowering): Medium; 65-75 days after emergence, which is similar to Moravian 37 and Moravian 69.
  • Plant Height Short; averages 73-84cm; 2-5 cm shorter than Moravian 69
  • Leaf Sheath Pubescence Absent Leaf Color at Boot: Green
  • Length Less than one-half of lemma
  • Kernel Aleurone Color Colorless
  • Kernel Weight 46-49g equal to Moravian 37 and Moravian 69
  • Moravian 115 is consistently 0.5-2.0 percent higher in malt extract than Moravian 69.
  • This invention is also directed to methods for producing a barley variety by crossing a first parent barley variety with a second parent barley variety, wherein the first or second barley variety is the variety V115. Therefore, any methods using the barley variety MV115 are part of this invention including selfing, backcrosses, hybrid breeding, and crosses to populations. Any plants produced using barley variety MV115 as a parent are within the scope of this invention.
  • Additional methods include, but are not limited to, expression vectors introduced into plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably, expression vectors are introduced into plant tissues by using either microprojectile-mediated delivery with a biolistic device or by using Agrobacterium-mediated transformation. Transformed plants obtained with the protoplasm of the invention are intended to be within the scope of this invention.
  • Further reproduction of the barley variety MV115 can occur by tissue culture and regeneration. Tissue culture of various tissues of barley and regeneration of plants therefrom is well known and widely published. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce barley plants capable of having the physiological and morphological characteristics of barley variety MV115.
  • plant parts includes plant protoplasts, plant cell tissue cultures from which barley plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, pericarp, seed, flowers, florets, heads, spikes, leaves, roots, root tips, anthers, pistils and the like.
  • transgenes DNA sequences, whether from a different species or from the same species, which are introduced into the genome using transformation or various breeding methods, are referred to herein collectively as "transgenes”.
  • a transgenic variant of MV115 may contain at least one transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.
  • Nucleic acids or polynucleotides refer to K A or DNA that is linear or branched, single or double stranded, or a hybrid thereof.
  • the term also encompasses RNA/DNA hybrids. These terms also encompass untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene.
  • Less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing.
  • polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
  • the antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides.
  • the polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR and in vitro or in vivo transcription.
  • One embodiment of the invention is a process for producing barley variety MVl 15 further comprising a desired trait, said process comprising introducing a gene that confers a desired trait to a barley plant of variety MVl 15.
  • Another embodiment is the product produced by this process.
  • the desired trait may be one or more of herbicide resistance, insect resistance, disease resistance, or modified fatty acid, carbohydrate or protein metabolism.
  • the specific gene may be any known in the art or listed herein, including; a polynucleotide conferring resistance to imidazolinone, dicamba, sulfonylurea, glyphosate, glufosinate, triazine, benzonitrile, cyclohexanedione, phenoxy proprionic acid and L-phosphinothricin; a polynucleotide encoding a Bacillus
  • thuringiensis polypeptide FAD-2, FAD-3, galactinol synthase or a raffinose synthetic enzyme, a nucleic acid molecule modifying protein metabolism, or a polynucleotide conferring resistance to rust, smut, BYDV or any other barley disease or pest.
  • Such a vector comprises a DNA sequence that contains a gene under the control of or operatively linked to a regulatory element, for example a promoter.
  • the vector may contain one or more genes and one or more regulatory elements.
  • a genetic trait which has been engineered into a particular barley plant using transformation techniques could be moved into another line using traditional breeding techniques that are well known in the plant breeding arts.
  • a backcrossing approach could be used to move a transgene from a transformed barley plant to an elite barley variety and the resulting progeny would comprise a transgene.
  • crossing can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
  • breeding cross excludes the processes of selfing or sibbing.
  • Various genetic elements can be introduced into the plant genome using transformation. These elements include but are not limited to genes, coding sequences, inducible, constitutive, and tissue specific promoters, enhancing sequences and signal and targeting sequences.
  • Plant transformation involves the construction of an expression vector that will function in plant cells.
  • a vector comprises DNA comprising a gene under control of, or operatively linked to, a regulatory element (for example, a promoter).
  • the expression vector may contain one or more such operably linked gene/regulatory element combinations.
  • the vector(s) may be in the form of a plasmid and can be used alone or in combination with other plasmids to provide transformed barley plants using
  • Expression vectors include at least one genetic marker operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker.
  • a regulatory element a promoter, for example
  • Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.
  • One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase ⁇ ( ⁇ ) gene which, when under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983).
  • Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al, Plant Mol. Biol, 5:299 (1985).
  • Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase and aminoglycoside-3'-adenyl transferase, the bleomycin resistance determinant (Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al. Plant Mol. Biol. 14:197 (1990), Hille et al. Plant Mol. Biol. 7:171 (1986)).
  • selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil (Comai et al. Nature 317:741-744 (1985), Gordon-Kamm et al. Plant Cell 2:603-618 (1990) and Stalker et al. Science 242:419-423 (1988)).
  • Selectable marker genes for plant transformation not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshildmate-3-phosphate synthase and plant acetolactate synthase (Eichholtz et al. Somatic Cell Mol. Genet. 13:67 (1987), Shah et al. Science 233:478 (1986), Charest et al. Plant Cell Rep. 8:643 (1990)).
  • Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include ⁇ -glucuronidase (GUS), ⁇ - galactosidase, luciferase and chloramphenicol acetyltransferase (Jefferson, R. A, Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al, EMBO J.
  • GUS ⁇ -glucuronidase
  • luciferase luciferase
  • chloramphenicol acetyltransferase Jefferson, R. A, Plant Mol. Biol. Rep. 5:387 (1987),
  • GFP Green Fluorescent Protein
  • Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter.
  • a regulatory element for example, a promoter.
  • Several types of promoters are well known in the transformation arts as are other regulatory elements that can be used alone or in combination with promoters.
  • promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred”.
  • tissue-specific Promoters that initiate transcription only in a certain tissue are referred to as "tissue-specific.”
  • a "cell-type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light.
  • Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters.
  • a “constitutive” promoter is a promoter that is active under most environmental conditions.
  • A. Inducible Promoters An inducible promoter is operably linked to a gene for expression in barley. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in barley. With an inducible promoter the rate of transcription increases in response to an inducing agent.
  • any inducible promoter can be used in the instant invention. See Ward et al, Plant Mol. Biol. 22:361-366 (1993).
  • Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993)); In2 gene from maize which responds to benzene sulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen.
  • a particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond.
  • An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci. USA 88:0421 (1991)).
  • constitutive Promoters - A constitutive promoter is operably linked to a gene for expression in barley or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in barley.
  • constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985)) and the promoters from such genes as rice actin (McElroy et al., Plant Cell 2: 163-171
  • ALS promoter Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xbal/Ncol fragment), represents a particularly useful constitutive promoter. See PCT Publication No. WO 96/30530.
  • tissue-specific or Tissue-preferred Promoters A tissue-specific promoter is operably linked to a gene for expression in barley. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence that is operably linked to a gene for expression in barley. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue. [0105] Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention.
  • tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter such as that from the phaseolin gene (Murai et al, Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(ll):2723-2729 (1985) and Timko et al, Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al, Mol.
  • a root-preferred promoter such as that from the phaseolin gene (Murai et al, Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.
  • a pollen-specific promoter such as that from Zml3 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).
  • Transport of a protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5' and/or 3' region of a gene encoding the protein of interest.
  • Targeting sequences at the 5' and/or 3' end of the structural gene may determine during protein synthesis and processing where the encoded protein is ultimately compartmentalized.
  • a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast.
  • Many signal sequences are known in the art. See, for example, Becker et al, Plant Mol. Biol..20:49 (1992); Knox, C, et al., Plant Mol. Biol. 9:3-17 (1987); Lerner et al, Plant Physiol.
  • transgenic plants With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities.
  • techniques for the selection and propagation of transformed plants which are well understood in the art, yield a plurality of transgenic plants that are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).
  • the transgenic plant provided for commercial production of foreign protein is a barley plant.
  • the biomass of interest is seed.
  • a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule.
  • RFLP radio frequency polymerase chain reaction
  • plants can be genetically engineered to express various phenotypes of agronomic interest.
  • genes can be altered to enhance disease resistance, insect resistance, herbicide resistance, agronomic, grain quality and other traits. Transformation can also be used to insert DNA sequences which control or help control male-sterility.
  • DNA sequences native to barley as well as non-native DNA sequences can be transformed into barley and used to alter levels of native or non-native proteins.
  • Various promoters, targeting sequences, enhancing sequences, and other DNA sequences can be inserted into the genome for the purpose of altering the expression of proteins. Reduction of the activity of specific genes (also known as gene silencing, or gene suppression) is desirable for several aspects of genetic engineering in plants.
  • knock-outs such as by insertion of a transposable element such as mu (Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Lox or other site specific integration site, antisense technology (see, e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.
  • WO 03/076574 and WO 99/25853 Zn-fmger targeted molecules (e.g., WO 01/52620;
  • agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Through the transformation of barley the expression of genes can be modulated to enhance disease resistance, insect resistance, herbicide resistance, water stress tolerance and agronomic traits as well as grain quality traits. Transformation can also be used to insert DNA sequences which control or help control male-sterility. DNA sequences native to barley as well as non-native DNA sequences can be transformed into barley and used to modulate levels of native or non- native proteins.
  • Anti-sense technology various promoters, targeting sequences, enhancing sequences, and other DNA sequences can be inserted into the barley genome for the purpose of modulating the expression of proteins.
  • Exemplary genes implicated in this regard include but are not limited to, those categorized below.
  • R disease resistance gene
  • Avr avirulence gene
  • a plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium folvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.
  • Fusarium head blight along with deoxynivalenol both produced by the pathogen Fusarium graminearum Schwabe have caused devastating losses in barley production.
  • Genes expressing proteins with antifungal action can be used as transgenes to prevent Fusarium head blight.
  • Various classes of proteins have been identified. Examples include endochitinases, exochitinases, glucanases, thionins, thaumatin-like proteins, osmotins, ribosome inactivating proteins, fiavonoids, and lactoferricin.
  • endochitinases include exochitinases, exochitinases, glucanases, thionins, thaumatin-like proteins, osmotins, ribosome inactivating proteins, fiavonoids, and lactoferricin.
  • Genes used to help reduce Fusarium head blight include but are not limited to Tril 01 (Fusarium), PDR5 (yeast), tip-l(oat), tlp-2(oat), leaf tip- 1 (wheat), tip (rice), tlp-4 (oat), endochitinase, exochitinase, glucanase (Fusarium), permatin (oat), seed hordothionin (barley), alpha-thionin (wheat), acid glucanase (alfalfa), chitinase (barley and rice), class beta ⁇ - 1,3 -glucanase (barley), PR5/tlp (Arabidopsis), zeamatin (maize), type 1 RTF (barley), NPR1 (Arabidopsis), lactoferrin (mammal), oxalyl-CoA-decarboxylase
  • (B) A gene conferring resistance to a pest, such as Hessian fly, wheat stem soft fly, cereal leaf beetle, and/or green bug.
  • a pest such as Hessian fly, wheat stem soft fly, cereal leaf beetle, and/or green bug.
  • a pest such as Hessian fly, wheat stem soft fly, cereal leaf beetle, and/or green bug.
  • H9, H10, and H21 genes are examples of the H9, H10, and H21 genes.
  • (C) A gene conferring resistance to such diseases as barley rusts, Septoria tritici, Septoria nodorum, powdery mildew, Helminthosporium diseases, smuts, bunts, Fusarium diseases, bacterial diseases, and viral diseases.
  • (D) A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications and hereby are incorporated by reference for this purpose: U.S. Pat. Nos.
  • (T) A molecule that stimulates signal transduction.
  • T A molecule that stimulates signal transduction.
  • Botella et al. Plant Molec. Biol. 24: 757 (1994)
  • nucleotide sequences for mung bean calmodulin cDNA clones and Griess et al., Plant Physiol. 104: 1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.
  • (L) A viral-invasive protein or a complex toxin derived therefrom.
  • the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses.
  • Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
  • (N) A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
  • (O) A developmental-arrestive protein produced in nature by a pathogen or a parasite.
  • fungal endo-alpha-l,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha-l,4-D-galacturonase.
  • the cloning and characterization of a gene which encodes a bean endo-poly-galacturonase-inhibiting protein is described by Toubart et al., Plant J. 2: 367 (1992).
  • (P) A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10: 305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
  • (S) Detoxification genes such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives. For example, see U.S. Pat. No. 5,792,931.
  • Other genes that confer tolerance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, and genes for various phosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619).
  • Exemplary genes in this category code for mutant ALS and ABAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449 (1990), respectively. See also, U.S. Pat. Nos.
  • PAT phosphinothricin acetyl transferase
  • bar Streptomyces hygroscopicus PAT
  • pyridinoxy or phenoxy proprionic acids and cycloshexones pyridinoxy or phenoxy proprionic acids and cycloshexones
  • Glyphosate resistance is also imparted to plants that express a gene that encodes a glyphosate oxido-reductase enzyme as described more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose.
  • glyphosate resistance can be imparted to plants by the over-expression of genes encoding glyphosate N-acetyltransferase.
  • ADNA molecule encoding a mutant aroA gene can be obtained under ATCC accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.
  • European Patent Application No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin.
  • nucleotide sequence of a PAT gene is provided in European Patent No. 0242 246 and 0 242236 to Leemans et al. De Greef et al., Bio/Technology 7: 61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for PAT activity. See also, U.S. Pat. Nos.
  • (D) An herbicide that inhibits photosynthesis such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene).
  • Przibilla et al. Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al, Biochem. J.285:173 (1992).
  • Protoporphyrinogen oxidase is necessary for the production of chlorophyll, which is necessary for all plant survival.
  • the protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Pat. Nos. 6,288,306 Bl; 6,282,837 Bl; and 5,767,373; and international publication WO 01/12825.
  • down-regulation of stearyl-ACP desaturase to increase stearic acid content of the plant, by for example, transforming a plant with a nucleic acid encoding an anti-sense of stearyl-ACP desaturase.
  • (B) Altered phosphorus content for example, the (1) Introduction of aphytase- encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127: 87 (1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene. (2) Up- regulation of a gene that reduces phytate content.
  • fatty acid modification genes mentioned above may also be used to affect starch content and/or composition through the interrelationship of the starch and oil pathways.
  • D Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols.
  • 5,432,068 describe a system of nuclear male sterility which includes: identifying a gene which is critical to male fertility; silencing this native gene which is critical to male fertility; removing the native promoter from the essential male fertility gene and replacing it with an inducible promoter; inserting this genetically engineered gene back into the plant; and thus creating a plant that is male sterile because the inducible promoter is not "on” resulting in the male fertility gene not being transcribed. Fertility is restored by inducing, or turning "on", the promoter, which in turn allows the gene that confers male fertility to be transcribed.
  • Other systems that may be used include the Gin recombinase of phage Mu (Maeser et al., 1991; Vicki Chandler, The Maize Handbook chapter 118 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto et al., 1983), and the R/RS system of the pSRl plasmid (Araki et al., 1992).
  • (A) may include but are not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance and increased yield under stress.
  • WO 00/73475 where water use efficiency is altered through alteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat. No. 6,717,034, U.S. Pat. No. 6,801,104, WO2000060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO
  • the HVA1 protein belongs to the group 3 LEA proteins that include other members such as wheat pMA2005 (Curry et al, 1991; Curry and Walker-Simmons, 1993), cotton D-7 (Baker et al., 1988), carrot Dc3 (Seffens et al., 1990), and rape pLEA76 (Harada et al., 1989).
  • Root and leaf tissues from transgenic plants regenerated from these transformed tobacco cells contained up to 100 mM mannitol.
  • Control plants contained no detectable mannitol.
  • Tarczynski et al. compared the growth of transgenic plants to that of untransformed control plants in the presence of 250 mM NaCl. After 30 days of exposure to 250 mM NaCl, transgenic plants had decreased weight loss and increased height relative to their untransformed counterparts. The authors concluded that the presence of mannitol in these transformed tobacco plants contributed to water deficit tolerance at the cellular level. See also U.S. Pat. No. 5,780,709 and international publication WO 92/19731 which are incorporated herein by reference for this purpose.
  • lipoxygenase levels can be altered to improve disease resistance (Steiner-Lange, S., et al. 2003. MPMI. 16(10):893-902.
  • Differential defense reactions in leaf tissues of barley in response to infection by Rh nchosporium secalis and to treatment with a fungal avirulence gene product and/or to improve the quality of the grain resulting in improved flavor for beer, cereal and other food products made from the grain (Douma, A., et al. 2003. U.S. Pat. No. 6,660,915).
  • beta-glucanase for improved plant and/or grain quality
  • Mutation breeding is another method of introducing new traits into barley variety MV115. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.
  • a further embodiment of the invention is a backcross conversion of barley variety MV11 .
  • a backcross conversion occurs when DNA sequences are introduced through traditional (non-transformation) breeding techniques, such as backcrossing. DNA sequences, whether naturally occurring or transgenes, may be introduced using these traditional breeding techniques. Desired traits transferred through this process include, but are not limited to nutritional enhancements, industrial enhancements, disease resistance, insect resistance, herbicide resistance, agronomic enhancements, grain quality
  • genes for other traits include: Leaf rust resistance genes (Lr series such as Lrl, LrlO, Lr21, Lr22, Lr22a, Lr32, Lr37, Lr41, Lr42, and Lr43), Fusarium head blight-resistance genes (QFhs.ndsu-3B and QFhs.ndsu-2A), Powdery Mildew resistance genes (Pm21), common bunt resistance genes (Bt-10), and wheat streak mosaic virus resistance gene (Wsml), Russian wheat aphid resistance genes (Dn series such as Dnl, Dn2, Dn4, Dn5), Black stem rust resistance genes (Sr38), Yellow rust resistance genes (Yr series such as Yrl, YrSD, Yrsu, Yrl7, Yrl5, YrH52), Aluminum tolerance genes (Alt(BH)), dwarf genes (Rht), vernalization genes (Vrn), Hessian fly resistance genes (H9, Leaf
  • the trait of interest is transferred from the donor parent to the recurrent parent, in this case, the barley plant disclosed herein.
  • Single gene traits may result from either the transfer of a dominant allele or a recessive allele.
  • Selection of progeny containing the trait of interest is done by direct selection for a trait associated with a dominant allele.
  • Selection of progeny for a trait that is transferred via a recessive allele requires growing and selfing the first backcross to determine which plants carry the recessive alleles.
  • Recessive traits may require additional progeny testing in successive backcross generations to determine the presence of the gene of interest.
  • Another embodiment of this invention is a method of developing a backcross conversion MVl 15 barley plant that involves the repeated backcrossing to barley variety MV115.
  • the number of backcrosses made may be 2, 3, 4, 5, 6 or greater, and the specific number of backcrosses used will depend upon the genetics of the donor parent and whether molecular markers are utilized in the backcrossing program. See, for example, von Bothmer, R. et al. 2003. Diversity in Barley (Elsevier Science) and Slafer, G. et al. 2002. Barley Science: Recent Advances from Molecular Biology to Agronomy of Yield and Quality (Haworth Press).
  • backcrossing methods one of ordinary skill in the art can develop individual plants and populations of plants that retain at least 70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the genetic profile of barley variety MV115.
  • the percentage of the genetics retained in the backcross conversion may be measured by either pedigree analysis or through the use of genetic techniques such as molecular markers or electrophoresis.
  • the backcross conversion developed from this method may be similar to MV115 for the results listed in Table 1. Such similarity may be measured by a side by side phenotypic comparison, with differences and similarities determined at a 5% significance level. Any such comparison should be made in environmental conditions that account for the trait being transferred. For example, herbicide should not be applied in the phenotypic comparison of herbicide resistant backcross conversion of MV115 to MV115.
  • Another embodiment of the invention is an essentially derived variety of MV115.
  • essentially derived varieties may be obtained for example by the selection of a natural or induced mutant, or of a somaclonal variant, the selection of a variant individual from plants of the initial variety, backcrossing, or transformation by genetic engineering.
  • An essentially derived variety of MV115 is further defined as one whose production requires the repeated use of variety MV115 or is predominately derived from variety MV115.
  • This invention also is directed to methods for using barley variety MV115 in plant breeding.
  • One such embodiment is the method of crossing barley variety MV115 with another variety of barley to form a first generation population of Fi plants.
  • the population of first generation Fi plants produced by this method is also an embodiment of the invention.
  • This first generation population of Fi plants will comprise an essentially complete set of the alleles of barley variety MV115.
  • One of ordinary skill in the art can utilize either breeder books or molecular methods to identify a particular Fi plant produced using barley variety MV115, and any such individual plant is also encompassed by this invention.
  • These embodiments also cover use of transgenic or backcross conversions of barley variety MV115 to produce first generation Fi plants.
  • a method of developing a MV115-progeny barley plant comprising crossing MVl 15 with a second barley plant and performing a breeding method is also an embodiment of the invention.
  • a specific method for producing a line derived from barley variety MVl 15 is as follows.
  • One of ordinary skill in the art would cross barley variety MVl 15 with another variety of barley, such as an elite variety.
  • the Fi seed derived from this cross would be grown to form a homogeneous population.
  • the Fi seed would contain one set of the alleles from variety MVl 15 and one set of the alleles from the other barley variety.
  • the Fi genome would be made-up of 50% variety MVl 15 and 50% of the other elite variety.
  • the Fi seed would be grown and allowed to self, thereby forming F 2 seed.
  • the F 2 seed would have derived 50% of its alleles from variety MVl 15 and 50% from the other barley variety, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15 (Wang J. and R. Bernardo, 2000, Crop Sci. 40:659-665 and Bernardo, R. and A. L. Kahler, 2001, Theor. Appl. Genet 102:986-992).
  • the F 2 seed would be grown and selection of plants would be made based on visual observation and/or measurement of traits.
  • the MVl 15 -derived progeny that exhibit one or more of the desired MVl 15-derived traits would be selected and each plant would be harvested separately. This F3 seed from each plant would be grown in individual rows and allowed to self. Then selected rows or plants from the rows would be harvested and threshed individually. The selections would again be based on visual observation and/or measurements for desirable traits of the plants, such as one or more of the desirable MVl 15-derived traits. The process of growing and selection would be repeated any number of times until a homozygous MVl 15-derived barley plant is obtained.
  • the homozygous MVl 15-derived barley plant would contain desirable traits derived from barley variety MVl 15, some of which may not have been expressed by the other original barley variety to which barley variety MVl 15 was crossed and some of which may have been expressed by both barley varieties but now would be at a level equal to or greater than the level expressed in barley variety MVl 15.
  • the homozygous MVl 15- derived barley plants would have, on average, 50% of their genes derived from barley variety MVl 15, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15.
  • the breeding process, of crossing, selfing, and selection may be repeated to produce another population of MVl 15-derived barley plants with, on average, 25% of their genes derived from barley variety MVl 15, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15.
  • Another embodiment of the invention is a homozygous MVl 15-derived barley plant that has received MVl 15-derived traits.
  • the previous example can be modified in numerous ways, for instance selection may or may not occur at every selfing generation, selection may occur before or after the actual self-pollination process occurs, or individual selections may be made by harvesting individual spikes, plants, rows or plots at any point during the breeding process described.
  • double haploid breeding methods may be used at any step in the process.
  • the population of plants produced at each and any generation of selfmg is also an embodiment of the invention, and each such population would consist of plants containing
  • Another embodiment of this invention is the method of obtaining a homozygous MV115-derived barley plant by crossing barley variety MV115 with another variety of barley and applying double haploid methods to the Fi seed or F] plant or to any generation of MV115-derived barley obtained by the selfing of this cross.
  • this invention also is directed to methods for producing MV115- derived barley plants by crossing barley variety MV115 with a barley plant and growing the progeny seed, and repeating the crossing or selfing along with the growing steps with the MV115-derived barley plant from 1 to 2 times, 1 to 3 times, 1 to 4 times, or 1 to .5 times.
  • any and all methods using barley variety MVT15 in breeding are part of this invention, including selfing, pedigree breeding, backcrossing, hybrid production and crosses to populations.
  • Unique starch profiles, molecular marker profiles and/or breeding records can be used by those of ordinary skill in the art to identify the progeny lines or populations derived from these breeding methods.
  • this invention also encompasses progeny with the same or greater yield or test weight of MV115, the same or shorter plant height, and the same or greater resistance to smut, stem rust, Septoria, net and spot blotch of MV115.
  • the expression of these traits may be measured by a side by side phenotypic comparison, with differences and similarities determined at a 5% significance level. Any such comparison should be made in the same environmental conditions.
  • barley cultivar MV115 was tested for malt protein production when grown in plots in one environment in Idaho in 2008, 2009 and 2010. Comparisons between MV115 and two Moravian cultivars, Moravian 037 and Moravian 069, are shown in Table 2 (2008 data) Table 3 (2009 data), Table 4 (2010 data). Table 2
  • a deposit of the MillerCoors proprietary barley cultivar designated MV115 disclosed above and recited in the appended claims will be made under the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Upon allowance of any claims i this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to the deposit with the American Type Culture Collection, Manassas, Va. The deposit will be maintained in the depository for a period of 30 . years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.

Abstract

A barley cultivar, designated MV115, is disclosed. MV115 is a high yield, lodging resistant cultivar with exceptional malting characteristics particularly useful in the brewing industry. The disclosure relates to seeds, plants, and to methods for producing a barley plant produced by crossing barley cultivar MV115 with itself or another barley variety. Methods for producing a barley plant containing in its genetic material one or more transgenes are disclosed. Barley varieties or breeding varieties, plant parts, methods for producing other barley varieties, lines or plant parts, and to the barley plants, varieties, and their parts derived from the use of those methods are disclosed. The disclosure further relates to hybrid barley seeds and plants produced by crossing bariey cultivar MV115 with another barley cultivar. Methods for developing other barley varieties or breeding lines derived from variety MV115 including cell and tissue culture, haploid systems, mutagenesis, and transgenic derived lines are disclosed.

Description

BARLEY CULTIVAR MORAVIAN 115
BACKGROUND OF INVENTION
[0001] The present invention relates to a new and distinctive barley cultivar designated Moravian 115 (MVl 15). All publications cited in this application are herein incorporated by reference.
[0002] Barley (Hordeum vulgare L.) is a grain that is grown worldwide with three main market classes, malt, feed and food. Most of the barley grain produced in the United States is used as an ingredient in cattle, pig, or poultry feed. Another major use for barley is malt production. Malt is used in the brewing and distilling industries to produce alcoholic beverages. Barley varieties that are preferred for producing malt are selected on the basis of characteristics such as kernel plumpness, low protein content and low Beta-glucan content. Barley grain that has more than about 13.5 weight percent protein on a dry basis or is too dark in color is rejected by malting plants. Significant overlap between the classes can occur since barley that does not meet malting specifications can be used for feed, food and potentially the emerging biofuels industry.
[0003] Barley is a nutritious food ingredient for humans or household pets. When used as a food ingredient, malting or feed barley grain that has a cemented hull (referred to as covered) must be processed to remove that hull. A commonly used processing step known as pearling removes the hull and a substantial portion of the bran and the germ to produce a pearled barley grain, such that at least about 15 to about 40 weight percent of the outer grain is removed. Barley varieties developed especially for food are hulless, i.e., they have a loose hull so do not have to be pearled prior to consumption. Hulless barley must be cleaned as do all grains prior to entering the human food markets, but loose hulls can be removed easily with only slight modifications to the cleaning plants. Food ingredient manufacturers may grind the cleaned barley to produce flour or roll the barley to produce flakes. Food ingredient manufacturers may also utilize the cleaned barley as a whole berry (seed).
[0004] Waxy barley is a naturally occurring variant that has recently been investigated for potential in food and industrial processing. Barley lines having the waxy phenotype have reduced amounts of amylose starch in the seed. The waxy trait may be useful in the production of high maltose syrup from barley (U.S. Pat. No. 4,116,770, Goering 1978) and in the production of flour and flakes (U.S. Pat. No. 5,614,242, Fox 1997 and U.S. Pat. No. 6,238,719, Fox, 2001) that have health benefits. [0005] The health promoting benefits of barley consumption have been investigated in human clinical trials. Studies have shown that individuals consuming barley that contains Beta-glucan soluble fiber have significant reductions in total and LDL plasma cholesterol (Behall et al. 2004. Am. J. Clin. Nutr. 80:1185-1193; Behall et al. 2004. J. Amer. Coll. Nutr.23:55-62) as well as blood pressure (Hallfrisch et al. 2003. Cer. Chem. 80:80-83; Behall et al. 2006. Nutrition. Res. 26:644-650). In May 2006, the FDA granted a petition to allow foods containing barley with 0.75 g of Beta-glucan to carry a health claim "barley lowers cholesterol when consumed as part of a healthy diet" (Federal Register
71(98):29248-29250).
[0006] Cultivated barley is a naturally self-fertilizing species, although there is a small percentage of cross-fertilization. Natural genetic and cytoplasmic male sterility is available to use in breeding and in hybrid seed production. Using all of the tools available to a breeder, it is possible to develop pure lines that are uniform in growth habit, maturity, yield, and other qualitative and quantitative characteristics. These lines can be released as inbred varieties, as inbreds for hybrid barley, or as lines to be further manipulated in the development of new lines or varieties or that incorporate proprietary genetic material.
[0007] Barley varieties may differ from each other in one or more traits and can be classified and differentiated according to the specific traits they possess. For example, there are types of barley known as two-rowed and other types known as six-rowed, referring to the number and positioning of kernels on the spike. Barley lines also can be classified as spring barley or winter barley, referring to the growth habit, or by the adherence of hulls on the seed, or by the type of starch in the seed. There are, of course, many other traits, which differentiate the various lines. A discussion of breeding methods for developing barley lines and of some traits in barley can be found in Foster, A. E., Barley, pp. 83-125, and in Fehr, W. R., ed., Principles of Cultivar Development Vol. 2 Crop species. Macmillan, N.Y. (1987). Once a breeder has developed a pure line, it may be given a unique name and released as a cultivar under that name. While named cultivars are not necessarily pure lines (they could be a mixture of genotypes or even be a hybrid) presently, most named barley cultivars are pure lines.
[0008] The present invention relates to a new and distinctive barley variety, designated Moravian 115 (MV115), which has been the result of years of careful breeding and selection as part of a barley breeding program. There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety an improved combination of desirable traits from the parental germplasm. These important traits may include higher seed yield, resistance to diseases and insects, tolerance to drought and heat, better agronomic qualities and improved grain quality.
[0009] Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is sib-pollinated when individuals within the same family or line are used for pollination. A plant is cross-pollinated if the pollen comes from a flower on a different plant from a different family or line. The term cross- pollination herein does not include self-pollination or sib-pollination.
[0010] A cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two heterozygous plants each that differ at a number of gene loci will produce a population of plants that differ genetically and will not be uniform. Regardless of parentage, plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. The term "homozygous plant" is hereby defined as a plant with homozygous genes at 95% or more of its loci. The term "inbred" as used herein refers to a homozygous plant or a collection of homozygous plants.
[0011] Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of variety used commercially (e.g., Fi hybrid variety, pureline variety, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.
[0012] The complexity of inheritance influences choice of the breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self- pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination and the number of hybrid offspring from each successful cross.
[0013] Pedigree breeding is commonly used for the improvement of self-pollinating crops. Two parents that possess favorable, complementary traits are crossed to produce an Fi . An F2 population is produced by selfing or sibbing one or several FiS. Selection of the best individuals may begin in the F2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families can begin in the F4
generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F5, F6 and F7), the best lines or mixtures of
phenot pically similar lines are tested for potential release as new varieties.
[0014] Backcross breeding has been used to transfer genes for simply inherited, qualitative, traits from a donor parent into a desirable homozygous variety that is utilized as the recurrent parent. The source of the traits to be transferred is called the donor parent. After the initial cross, individuals possessing the desired trait or traits of the donor parent are selected and then repeatedly crossed (backcrossed) to the recurrent patent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., variety) plus the desirable trait or traits transferred from the donor parent. This approach has been used extensively for breeding disease resistant varieties.
[0015] Each barley breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful varieties produced per unit of input (e.g., per year, per dollar expended, etc.).
[0016] Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination and the number of hybrid offspring from each successful cross. Recurrent selection can be used to improve populations of either self- or cross-pollinated crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued. Plants from the populations can be selected and selfed to create new varieties. [0017] Another breeding method is single-seed descent. This procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F2 to the desired level of inbreeding, the plants from which lines are derived will each trace to different F2 individuals. The number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F2 plants originally sampled in the population will be represented by a progeny when generation advance has been completed. In a multiple-seed procedure, barley breeders commonly harvest one or more spikes
(heads) from each plant in a population and thresh them together to form a bulk. Part of the bulk is used to plant the next generation and part is put in reserve. The procedure has been referred to as modified single-seed descent. The multiple-seed procedure has been used to save labor at harvest. It is considerably faster to thresh spikes with a machine than to remove one seed from each by hand for the single-seed procedure. The multiple-seed procedure also makes it possible to plant the same number of seeds of a population each generation of inbreeding. Enough seeds are harvested to make up for those plants that did not germinate or produce seed.
[0018] Bulk breeding can also be used. In the bulk breeding method an F2 population is grown. The seed from the populations is harvested in bulk and a sample of the seed is used to make a planting the next season. This cycle can be repeated several times. In general when individual plants are expected to have a high degree of homozygosity, individual plants are selected, tested, and increased for possible use as a variety.
[0019] Molecular markers including techniques such as Starch Gel Electrophoresis, Isozyme Eletrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple
Sequence Repeats (SSRs), and Single Nucleotide Polymorphisms (SNPs) may be used in plant breeding methods. One use of molecular markers is Quantitative Trait Loci (QTL) mapping. QTL mapping is the use of markers, which are known to be closely linked to alleles that have measurable effects on a quantitative trait. Selection in the breeding process is based upon the accumulation of markers linked to the positive effecting alleles and/or the elimination of the markers linked to the negative effecting alleles from the plant's genome.
[0020] Molecular markers can also be used during the breeding process for the selection of qualitative traits. For example, markers closely linked to alleles or markers containing sequences within the actual alleles of interest can be used to select plants that contain the alleles of interest during a backcrossing breeding program. The markers can also be used to select for the genome of the recurrent parent and against the markers of the donor parent. Using this procedure can minimize the amount of genome from the donor parent that remains in the selected plants. It can also be used to reduce the number of crosses back to the recurrent parent needed in a backcrossing program (Openshaw et al. Marker- assisted Selection in Backcross Breeding. In: Proceedings Symposium of the Analysis of Molecular Marker Data, 5-6 Aug. 1994, pp. 41-43. Crop Science Society of America, Corvallis, Oreg.). The use of molecular markers in the selection process is often called Genetic Marker Enhanced Selection.
[0021] The production of double haploids can also be used for the development of homozygous lines in the breeding program. Double haploids are produced by the doubling of a set of chromosomes (IN) from . a heterozygous plant to produce a completely homozygous individual. This can be advantageous because the process omits the generations of selfing needed to obtain a homozygous plant from a heterozygous source. Various methodologies of making double haploid plants in barley have been developed (Laurie, D. A. and S. Reymondie, Plant Breeding, 1991, v. 106:182-189. Singh, N. et al, Cereal Research Communications, 2001, v. 29:289-296; Redha, A. et al., Plant Cell Tissue and Organ Culture, 2000, v. 63:167-172; U.S. Pat. No. 6,362,393)
[0022] Though pure-line varieties are the predorninate form of barley grown for
' commercial barley production hybrid barley is also used. Hybrid barleys are produced with the help of cytoplasmic male sterility, nuclear genetic male sterility, or chemicals. Various combinations of these three male sterility systems have been used in the production of hybrid barley.
[0023] Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., Allard, Principles of Plant Breeding, 1960; Simmonds, Principles of Crop Improvement, 1979).
[0024] Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s). The best lines are candidates for new commercial varieties; those still deficient in a few traits may be used as parents to produce new populations for further selection. [0025] A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior genotype is to observe its performance relative to other experimental genotypes and to a widely grown standard variety. Generally a single observation is inconclusive, so replicated observations are required to provide a better estimate of its genetic worth.
[0026] A breeder uses various methods to help determine which plants should be selected from the segregating populations and ultimately which lines will be used for
commercialization. In addition to the knowledge of the germplasm and other skills the breeder uses, a part of the selection process is dependent on experimental design coupled with the use of statistical analysis. Experimental design and statistical analysis are used to help determine which plants, which family of plants, and finally which lines are significantly better or different for one or more traits of interest. Experimental design methods are used to control error so that differences between two lines can be more accurately determined. Statistical analysis includes the calculation of mean values, determination of the statistical significance of the sources of variation, and the calculation of the appropriate variance components. Five and one percent significance levels are customarily used to determine whether a difference that occurs for a given trait is real or due to the environment or experimental error.
[0027] Plant breeding is the genetic manipulation of plants. The goal of barley breeding is to develop new, unique and superior barley varieties. In practical application of a barley breeding program, the breeder initially selects and crosses two or more parental lines, followed by repeated selfing and selection, producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via crossing, selection, selfing and mutations. Therefore, a breeder will never develop the same line, or even very similar lines, having the same barley traits from the exact same parents.
[0028] Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under unique and different geographical, climatic and soil conditions, and further selections are then made during and at the end of the growing season. The cultivars that are developed are unpredictable because the breeder's selection occurs in unique environments with no control at the DNA level, and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting lines he develops, except possibly in a very gross and general fashion. The same breeder cannot produce the same cultivar twice by using the same original parents and the same selection techniques. This unpredictability results in the expenditure of large amounts of research monies to develop superior new barley cultivars.
[0029] Proper testing should detect major faults and establish the level of superiority or improvement over current varieties. In addition to showing superior performance, there must be a demand for a new variety. The new variety must be compatible with industry standards, or must create a new market. The introduction of a new variety may incur additional costs to the seed producer, the grower, processor and consumer, for special advertising and marketing, altered seed and commercial production practices, and new product utilization. The testing preceding release of a new variety should take into consideration research and development costs as well as technical superiority of the final variety. It must also be feasible to produce seed easily and economically.
[0030] These processes, which lead to the final step of marketing and distribution, can take from six to twelve years from the time the first cross is made. Therefore, development of new varieties is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.
[0031] Barley is an important and valuable field crop. Thus, a continuing goal of barley breeders is to develop stable, high yielding barley varieties that are agronomically sound and have good grain quality for its intended use. To accomplish this goal, the barley breeder must select and develop barley plants that have the traits that result in superior varieties.
[0032] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.
SUMMARY OF INVENTION
[0033] The following embodiments and aspects thereof are described in conjunction with systems, tools, and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
[0034] According to the invention, there is provided a new barley cultivar designated Moravian 115 (MVl 15). This invention thus relates to the seeds of barley cultivar MVl 15, to the plants of barley cultivar MVl 15 and to methods for producing a barley plant produced by crossing the barley cultivar MV115 with itself or another barley cultivar, and the creation of variants by mutagenesis or transformation of barley cultivar MVl 15.
[0035] Thus, any such methods using the barley cultivar MVl 15 are part of this invention: selling, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using barley cultivar MVl 15 as at least one parent are within the scope of this invention. Advantageously, the barley cultivar could be used in crosses with other, different, barley plants to produce first generation (Fi) barley hybrid seeds and plants with superior characteristics.
[0036] In another aspect, the present invention provides for single or multiple gene converted plants of barley cultivar MVl 15. The transferred gene(s) may preferably be a dominant or recessive allele. Preferably, the transferred gene(s) will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, male sterility, enhanced nutritional quality, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified protein percent, modified beta-glucan percent, modified lodging resistance, modified lipoxygenase, beta- glucanase and/or polyphenol oxidase content and/or activity, and industrial usage. The gene may be a naturally occurring barley gene or a transgene introduced through genetic engineering techniques.
[0037] In another aspect, the present invention provides regenerable cells for use in tissue culture of barley plant MVl 15. The tissue culture will preferably be capable of regenerating plants having essentially all the physiological and morphological characteristics of the foregoing barley plant, and of regenerating plants having
substantially the same genotype as the foregoing barley plant. Preferably, the regenerable cells in such tissue cultures will be selected or produced from head, awn, leaf, pollen, embryo, cotyledon, hypocotyl, seed, spike, pericarp, meristematic cell, protoplast, root, root tip, pistil, anther, floret, shoot, stem and callus. Still further, the present invention provides barley plants regenerated from the tissue cultures of the invention.
[0038] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.
DEFINITIONS
[0039] AA Dry Basis: Alpha-amylase activity of malt per dry weight of malt. Alpha- amylase is an endogenous enzyme of barley that is produced during germination. It degrades malt starch to dextrins. It is also called liquefying enzyme as it reduces viscosity of gelatinized starch solution. [0040] Awn. Awn is intended to mean the elongated needle-like appendages on the flower-and seed-bearing "head" at the top of the barley plant. These awns are attached to the lemmas. Lemmas enclose the stamen and the stigma as part of the florets. These florets are grouped in spikelets, which in turn together comprise the head or spike.
[0041] Beta-glucan Dry Basis: beta-glucan is barley cell wall component that is degraded during germination to enable starch extraction from the grain. The beta-glucan content of malt indicates the completeness of cell wall modification. The lower the beta-glucan content, the more complete cell wall degradation is presumed. High beta-glucan content of malt can impede lautering and beer filtration.
[0042] Beta-glucan fiber. Beta-glucan fiber is a nonstarch polysaccharide in which individual glucose molecules (20,000-1,000,000) are linked by beta 1-4 and beta 1-3 linkages. Beta-glucan is soluble in warm water (40-45 degrees Centigrade); cellulose is insoluble in water. Beta-glucan is the main structural material in the cell walls of barley and oat grain.
[0043] Beta-glucan fiber viscosity. Beta-glucan fiber viscosity describes the friction that is created in a solution by the presence of beta-glucan chains (fibers) and is measured in centipoise units.
[0044] Centipoise units (cps). Centipoise units (cps) are the units commonly used to measure viscosity. By definition the fundamental unit of viscosity measurement is the "Poise", which is a material requiring a sheer stress of one dyne per square centimeter to produce a sheer of one inverse second, which has a viscosity of one poise or 100 centipoise.
[0045] Covered seed. Barley seed can have a cutin layer which cements the hull (lemma and palea or glumes) to the seed. This trait is controlled by the Nud locus on chromosome 1 (7H). The homozygous dominant Nud Nud genotype results in the presence of cutin and is referred to as covered. The hull can only be removed by abrasive processing prior to consumption, known as pearling.
[0046] DP Dry Basis: Diastatic Power (DP) value indicates the malt enzyme activity that degrades dextrins and starch to fermentable sugars.
[0047] Extract Dry Basis: Extract value indicates the percentage of dry malt matter that can be solubilized to water during mashing. It is determined by measuring specific gravity of wort.
[0048] Essentially all of the physiological and morphological characteristics. A plant having essentially all of the physiological and morphological characteristics means a plant having the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted trait.
[0049] FAN Dry Basis: Free Amino Nitrogen (FAN) consists of free amino acids and small peptides. They are produced from barley proteins by proteolytic eruzymes during malting and mashing.
[0050] Foliar disease: Foliar disease is a general term for fungal disease which causes yellowing or browning or premature drying of the leaves. The disease typically involves Septoria, net blotch, spot blotch or scald.
[0051] Gene. As used herein, "gene" refers to a segment of nucleic acid. A gene can be introduced into a genome of a species, whether from a different species or from the same species, using transformation or various breeding methods.
[0052] Head. As used herein, the term "head" refers to a group of spikelets at the top of one plant stem. The term "spike" also refers to the head of a barley plant located at the top of one plant stem.
[0053] Lodging. As used herein, the term "lodging" refers to the bending or breakage of the plant stem, or the tilting over of the plant, which complicates harvest and can diminish the value of the harvested product.
[0054] Leaf rust: A fungal disease that results in orange-red pustules on the leaf surface. Caused by Puccinia hordei.
[0055] Malt Protein: Total protein content of malt. Typically 40-50% of the malt protein is soluble in wort.
[0056] Maturity (HEB): Maturity, higher is better (FEB) refers to the physiological stage of plant growth used to describe early or lateness of the variety. Maturity is measured on a scale of 3- 1. The higher the maturity number the earlier maturing the variety.
[0057] Net blotch: Net blotch refers to a fungal disease which appears as elongated black lesions running parallel to the leaf veins with distinctive, dark brown net-like patterns. Net blotch is caused by Pyrenophora teres.
[0058] Plant. As used herein, the term "plant" includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. A seed or embryo that will produce the plant is also considered to be the plant.
[0059] Plant height. As used herein, the term "plant height" is defined as the average height in inches or centimeters of a group of plants, as measured from the ground level to the tip of the head, excluding awns. [0060] Plant parts. As used herein, the term "plant parts" (or a barley plant, or a part thereof) includes but is not limited to protoplasts, leaves, stems, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, flower, shoot, tissue, petiole, cells, meristematic cells, head, awn, spike, pericarp, pistil, and callus and the like.
[0061] Plump (%): Refers to the percentage of grain retained on a 6/64" screen when shaken on a timed mechanical shaker for 15 revolutions. This test measures the fraction of kernels that are termed "plump grain".
[0062] Progeny. As used herein, progeny includes an barley plant produced from the cross of two barley plants where at least one plant includes barley cultivar Moravian 115 and progeny further includes but is not limited to subsequent F2, F3, F4, F5, F6, F7, Fg, F9 and F10 generational crosses with the recurrent parental line. Progeny also includes Si plant produced from the selfing of barley cultivar Moravian 115; progeny further includes but is not limited to subsequent selfing generations and crosses with the recurrent parental line.
[0063] Regeneration. Regeneration refers to the development of a plant from tissue culture.
[0064] Scald: Scald refers to a fungal disease that causes spots to develop on the leaves during cool, wet weather. The spots are oval shaped and the margins of the spots change from bluish-green to zonated brown or tan rings with bleached straw-colored centers. Scald is caused by Rkynchosporium secalis.
[0065] Septoria: Septoria refers to a fungal disease that appears as elongated, light brown spots on the leaves. It is caused by Septoria passerinii.
[0066] Single gene converted (Conversion). Single gene converted (conversion) plants refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique, genetic engineering or mutation, either induced or spontaneous.
[0067] Smut, covered: Covered smut refers to a fungal disease in which masses of black spores replace the seed kernels on the head. A persistent membrane can be ruptured during harvest to disperse spores. Covered smut is caused by Ustilago hordei.
Smut, loose: Loose smut refers to a fungal disease in which masses of black spores replace the seed kernels on the head. The thin membrane that covers the spores is easily ruptured and spores disbursed by wind. Loose smut is caused by Ustilago nuda. [0068] Spot blotch: Spot blotch refers to a fungal disease that appears as dark, chocolate- colored blotches forming irregular dead patches on the leaves. Spot blotch is caused by Cochliobolus sativus.
[0069] Stem rust: Stem rust refers to a fungal disease that produces masses of brick-red pustules on stems and leaf sheaths. Stem rust can be caused by either Puccinia graminis f. sp. tritici or Puccinia graminis f. sp. secalis.
[0070] Stripe rust: Stripe rust refers to a fungal disease that results in light yellowish orange pustules arranged in stripes between the veins of the leaves. Stripe rust is caused by Puccinia striiformis f. sp. hordei.
[0071] Test Weight: The test weight per bushel (U.S.) is the weight (in pounds) of the volume of grain required to fill level full a Winchester bushel measure of 2,150.42 cubic inches capacity" ( ef. 2, Chap. XI). This translates into pounds per 32 U.S. dry quarts or pounds per 35.238 L.
[0072] Viscosity: Resistance of fluid to flow. Wort viscosity indicates the presence of soluble dextrins, cell wall beta-glucans or gums in the wort that can impede lautering and beer filtration.
BREEDING INFORMATION
[0073] Moravian 115 (previously also known as CI 15) is a small-statured, high yielding, two-rowed malting barley with high extract, moderate protein (10.5-12.5%) and bright, plump seed that originated from the cross C97-73-05/C57. Both parents are high yielding malting lines.
[0074] The Fi was grown near Burley, Idaho in the winter of 2001 and 2002 and the F2 and F3 plants were greenhouse grown near Burley, Idaho in the spring and fall, respectively of 2002. Single F4 plants were selected in the summer of 2003 and one such plant was given the designation C01-47-14. This single plant was planted as an individual single row in a winter nursery near Leeston, New Zealand in 2003-2004. F4:F6 single, un-replicated plots were produced near Burley, Idaho in the spring and summer of 2004. Seed from this single plot was used for malting and for producing replicated plots in 2005. During subsequent increases and evaluations through the F6 to Fn generations, Moravian 115 proved to be stable and uniform. The Fs through Fn generations were tested for grain yield and other agronomic traits in trials from 2004 through 2010. Moravian 115 is a high yielding, lodging resistant cultivar with exceptional malting characteristics and resistance to rust fungus, that will be used in the brewing industry. DETAILED DESCRIPTION OF THE INVENTION
[0075] Moravian 115 is a semi-dwarf, two-rowed, spring habit malting barley variety selected from a breeding population for high yield and exceptional malting characteristics. Moravian 115 will be used as malting barley for the brewing industry.
[0076] Moravian 115 is a two-rowed, medium-maturing, short variety that has an early prostrate growth habit and has medium length awns that is adapted to the high desert areas of the Mountain States. Moravian 115 was selected for high agronomic performance and malting quality. The agronomic and malting characteristics of Moravian 115 are listed in Table 1. Comparisons between Moravian 115 and other Moravian barley lines are in Tables 2-5.
[0077] The spike of Moravian 115 is two-rowed, has a straight neck, a closed collar, is non-waxy, and erect at maturity. The spike has a few hairs on the rachis edge. The glumes of Moravian 115 are less than equal to the length of the glume, have a band of short hairs, and are awnless. The lemma has medium length awns that are rough. The base of the lemma has a depression and the rachilla has many long hairs. Seeds of Moravian 115 are hulled with a white aleurone, are midlong and have wrinkled, loosely attached hulls. The spike of Moravian 115 typically does not emerge completely from the flag leaf sheath or boot. Typically, Moravian 115 is most successfully produced under irrigated conditions and does not lodge under normal production conditions.
Table 1
VARIETY DESCRIPTION INFORMATION
Plant:
Growth Habit: Spring
Spike: Two-row
Juvenile Growth Habit: Prostrate
Plant Tillering: Intermediate
Maturity (50% flowering): Medium; 65-75 days after emergence, which is similar to Moravian 37 and Moravian 69.
Plant Height: Short; averages 73-84cm; 2-5 cm shorter than Moravian 69
Stem Color at Maturity: Yellow
Stem Strength: Medium
Lodging Resistant: Yes
Neck Shape: Straight
Collar Shape: Closed
Leaves:
Leaf Sheath Pubescence: Absent Leaf Color at Boot: Green
Flag Leaf at Boot: Upright, very petite
Pubescence on Leaf (first leaf below flag leaf) Blade: No
Pubescence on Leaf (first leaf below flag leaf) Sheath: No
Auricle Color: White
Pubescence on Auricle: Absent
Spike:
Density: Dense
Position at Maturity: Erect to partially nodding
Length of Spike: Intermediate
Hairiness of Rachis Edge: Few
Rachilla Hairs: Long
Lateral Florets: Sterile
Awns:
Awns: Straight
Length: Medium
Surface: Rough
Anthocyanin: Present
Glumes:
Length: Less than one-half of lemma
Hairiness: Banded
Length of Hairs: Short
Glume Awns: Absent
Glume Awn Length Relative to Glume Length: NA
Hull/Kernel:
Hull Type (Lemma Palea Adherence): Hulled
Hull: Wrinkled
Hairs on Ventral Furrow: Absent
Shape of Base: Depression
Kernel Aleurone Color: Colorless
Kernel Length: Mid
Kernel Weight: 46-49g equal to Moravian 37 and Moravian 69
Diseases:
Scald: Susceptible
Net and Spot blotch: Susceptible
Smut, loose and covered: Susceptible
Septoria: Not tested
Other Characteristics: Under commercial malting conditions, Moravian 115 is consistently 0.5-2.0 percent higher in malt extract than Moravian 69. [0078] This invention is also directed to methods for producing a barley variety by crossing a first parent barley variety with a second parent barley variety, wherein the first or second barley variety is the variety V115. Therefore, any methods using the barley variety MV115 are part of this invention including selfing, backcrosses, hybrid breeding, and crosses to populations. Any plants produced using barley variety MV115 as a parent are within the scope of this invention.
[0079] Additional methods include, but are not limited to, expression vectors introduced into plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably, expression vectors are introduced into plant tissues by using either microprojectile-mediated delivery with a biolistic device or by using Agrobacterium-mediated transformation. Transformed plants obtained with the protoplasm of the invention are intended to be within the scope of this invention.
[0080] Further reproduction of the barley variety MV115 can occur by tissue culture and regeneration. Tissue culture of various tissues of barley and regeneration of plants therefrom is well known and widely published. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce barley plants capable of having the physiological and morphological characteristics of barley variety MV115.
[0081] As used herein, the term plant parts includes plant protoplasts, plant cell tissue cultures from which barley plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, pericarp, seed, flowers, florets, heads, spikes, leaves, roots, root tips, anthers, pistils and the like.
Further Embodiments of the Invention
[0082] The advent of new molecular biological techniques has allowed the isolation and characterization of genetic elements with specific functions, such as encoding specific protein products. Scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and express foreign genetic elements, or additional, or modified versions of native or endogenous genetic elements in order to alter the traits of a plant in a specific manner. Any DNA sequences, whether from a different species or from the same species, which are introduced into the genome using transformation or various breeding methods, are referred to herein collectively as "transgenes". In some embodiments of the invention, a transgenic variant of MV115 may contain at least one transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, and the present invention also relates to transgenic variants of the claimed barley variety MVl 15.
[0083] Nucleic acids or polynucleotides refer to K A or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. These terms also encompass untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR and in vitro or in vivo transcription.
[0084] One embodiment of the invention is a process for producing barley variety MVl 15 further comprising a desired trait, said process comprising introducing a gene that confers a desired trait to a barley plant of variety MVl 15. Another embodiment is the product produced by this process. In one embodiment the desired trait may be one or more of herbicide resistance, insect resistance, disease resistance, or modified fatty acid, carbohydrate or protein metabolism. The specific gene may be any known in the art or listed herein, including; a polynucleotide conferring resistance to imidazolinone, dicamba, sulfonylurea, glyphosate, glufosinate, triazine, benzonitrile, cyclohexanedione, phenoxy proprionic acid and L-phosphinothricin; a polynucleotide encoding a Bacillus
thuringiensis polypeptide, FAD-2, FAD-3, galactinol synthase or a raffinose synthetic enzyme, a nucleic acid molecule modifying protein metabolism, or a polynucleotide conferring resistance to rust, smut, BYDV or any other barley disease or pest.
[0085] Numerous methods for plant transformation have been developed, including biological and physical plant transformation protocols. See, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al, "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.
[0086] The most prevalent types of plant transformation involve the construction of an expression vector. Such a vector comprises a DNA sequence that contains a gene under the control of or operatively linked to a regulatory element, for example a promoter. The vector may contain one or more genes and one or more regulatory elements.
[0087] A genetic trait which has been engineered into a particular barley plant using transformation techniques could be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move a transgene from a transformed barley plant to an elite barley variety and the resulting progeny would comprise a transgene. As used herein, "crossing" can refer to a simple X by Y cross, or the process of backcrossing, depending on the context. The term "breeding cross" excludes the processes of selfing or sibbing.
[0088] Various genetic elements can be introduced into the plant genome using transformation. These elements include but are not limited to genes, coding sequences, inducible, constitutive, and tissue specific promoters, enhancing sequences and signal and targeting sequences.
[0089] Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA comprising a gene under control of, or operatively linked to, a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid and can be used alone or in combination with other plasmids to provide transformed barley plants using
transformation methods as described below to incorporate transgenes into the genetic material of the barley plant(s).
Expression Vectors for Barley Transformation: Marker Genes
[0090] Expression vectors include at least one genetic marker operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.
[0091] One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase Π (ηρΐΠ) gene which, when under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al, Plant Mol. Biol, 5:299 (1985).
[0092] Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase and aminoglycoside-3'-adenyl transferase, the bleomycin resistance determinant (Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al. Plant Mol. Biol. 14:197 (1990), Hille et al. Plant Mol. Biol. 7:171 (1986)). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil (Comai et al. Nature 317:741-744 (1985), Gordon-Kamm et al. Plant Cell 2:603-618 (1990) and Stalker et al. Science 242:419-423 (1988)).
[0093] Selectable marker genes for plant transformation not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshildmate-3-phosphate synthase and plant acetolactate synthase (Eichholtz et al. Somatic Cell Mol. Genet. 13:67 (1987), Shah et al. Science 233:478 (1986), Charest et al. Plant Cell Rep. 8:643 (1990)).
[0095] Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β -glucuronidase (GUS), β- galactosidase, luciferase and chloramphenicol acetyltransferase (Jefferson, R. A, Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al, EMBO J. 8:343 (1989), Koncz et al, Proc. Natl. Acad. Sci. USA 84:131 (1987), DeBlock et al, EMBO J. 3:1681 (1984)). [0096] In vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available (Molecular Probes publication 2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115:151a (1991)). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.
[0097] More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFP may be used as screenable markers.
Expression Vectors for Barley Transformation: Promoters
[0098] Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are well known in the transformation arts as are other regulatory elements that can be used alone or in combination with promoters.
[0099] As used herein, "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred". Promoters that initiate transcription only in a certain tissue are referred to as "tissue-specific." A "cell-type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active under most environmental conditions.
[0100] A. Inducible Promoters—An inducible promoter is operably linked to a gene for expression in barley. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in barley. With an inducible promoter the rate of transcription increases in response to an inducing agent.
[0101] Any inducible promoter can be used in the instant invention. See Ward et al, Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993)); In2 gene from maize which responds to benzene sulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from TnlO (Gatz et al, Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci. USA 88:0421 (1991)).
[0102] B. Constitutive Promoters - A constitutive promoter is operably linked to a gene for expression in barley or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in barley.
[0103] Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985)) and the promoters from such genes as rice actin (McElroy et al., Plant Cell 2: 163-171
(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1 92)); pEMU (Last et al., Theor. Appl. Genet.
81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics 231 :276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xbal/Ncol fragment), represents a particularly useful constitutive promoter. See PCT Publication No. WO 96/30530.
[0104] C. Tissue-specific or Tissue-preferred Promoters—A tissue-specific promoter is operably linked to a gene for expression in barley. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence that is operably linked to a gene for expression in barley. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue. [0105] Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter such as that from the phaseolin gene (Murai et al, Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(ll):2723-2729 (1985) and Timko et al, Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al, Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zml3 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).
Signal Sequences for Targeting Proteins to Subcellular Compartments
[0106] Transport of a protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5' and/or 3' region of a gene encoding the protein of interest. Targeting sequences at the 5' and/or 3' end of the structural gene may determine during protein synthesis and processing where the encoded protein is ultimately compartmentalized.
[0107] The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example, Becker et al, Plant Mol. Biol..20:49 (1992); Knox, C, et al., Plant Mol. Biol. 9:3-17 (1987); Lerner et al, Plant Physiol.
91:124-129 (1989); Frontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al., Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al, J. Cell. Biol. 108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, et al., Cell 39:499-509 (1984); Steifel, et al, Plant Cell 2:785-793 (1990).
Foreign Protein Genes and Agronomic Genes
[0108] With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants that are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).
[0109] According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is a barley plant. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and
Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant.
[0110] Wang et al. discuss "Large Scale Identification, Mapping and Genotyping of Single-Nucleotide Polymorphisms in the Human Genome", Science, 280:1077-1082, 1998, and similar capabilities are becoming increasingly available for the barley genome. Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques. S Ps may also be used alone or in combination with other techniques.
[0111] Likewise, by means of the present invention, plants can be genetically engineered to express various phenotypes of agronomic interest. Through the transformation of barley the expression of genes can be altered to enhance disease resistance, insect resistance, herbicide resistance, agronomic, grain quality and other traits. Transformation can also be used to insert DNA sequences which control or help control male-sterility. DNA sequences native to barley as well as non-native DNA sequences can be transformed into barley and used to alter levels of native or non-native proteins. Various promoters, targeting sequences, enhancing sequences, and other DNA sequences can be inserted into the genome for the purpose of altering the expression of proteins. Reduction of the activity of specific genes (also known as gene silencing, or gene suppression) is desirable for several aspects of genetic engineering in plants.
[0112] Many techniques for gene silencing are well known to one of skill in the art, including but not limited to knock-outs (such as by insertion of a transposable element such as mu (Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Lox or other site specific integration site, antisense technology (see, e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.
5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor (1997) Plant Cell 9: 1245; Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA
91:3490-3496; Finnegan et al. (1994) Bio/Technology 12: 883-888; and Neuhuber et al.
(1994) Mol. Gen. Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell
2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore et al.
(2000) Cell 101:25-33; and Montgomery et al. (1998) PNAS USA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000) Plant Cell 12:691-705; and Baulcombe
(1999) Curr. Op. Plant Bio. 2: 109-113); target-RNA-specific ribozymes (Haseloff et al.
(1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000) Nature 407:319-320;
WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai (2003) Plant Cell
15:2730-2741); ribozymes (Steinecke et al. (1992) EMBO J. 11.T525; and Perriman et al. (1993) Antisense Res. Dev. 3 :253); oligonucleotide mediated targeted. modification (e.g.,
WO 03/076574 and WO 99/25853); Zn-fmger targeted molecules (e.g., WO 01/52620;
WO 03/048345; and WO 00/42219); and other methods or combinations of the above methods known to those of skill in the art.
[0113] Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Through the transformation of barley the expression of genes can be modulated to enhance disease resistance, insect resistance, herbicide resistance, water stress tolerance and agronomic traits as well as grain quality traits. Transformation can also be used to insert DNA sequences which control or help control male-sterility. DNA sequences native to barley as well as non-native DNA sequences can be transformed into barley and used to modulate levels of native or non- native proteins. Anti-sense technology, various promoters, targeting sequences, enhancing sequences, and other DNA sequences can be inserted into the barley genome for the purpose of modulating the expression of proteins. Exemplary genes implicated in this regard include but are not limited to, those categorized below.
1. Genes that Confer Resistance to Pests or Disease and that Encode:
[0114] (A) Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium folvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae); McDowell & Woffenden, (2003) Trends Biotechnol. 21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. ll(6):567-82.
[0115] Fusarium head blight along with deoxynivalenol both produced by the pathogen Fusarium graminearum Schwabe have caused devastating losses in barley production. Genes expressing proteins with antifungal action can be used as transgenes to prevent Fusarium head blight. Various classes of proteins have been identified. Examples include endochitinases, exochitinases, glucanases, thionins, thaumatin-like proteins, osmotins, ribosome inactivating proteins, fiavonoids, and lactoferricin. During infection with Fusarium graminearum deoxynivalenol is produced. There is evidence that production of deoxynivalenol increases the virulence of the disease. Genes with properties for detoxification of deoxynivalenol (Adam and Lemmens, In International Congress on Molecular Plant-Microbe Interactions, 1996; McCormick et al. Appl. Environ. Micro. 65:5252-5256, 1999) have been engineered for use in barley. A synthetic peptide that competes with deoxynivalenol has been identified (Y uan et al, Appl. Environ. Micro. 65:3279-3286 (1999)). Changing the ribosomes of the host so that they have reduced affinity for deoxynivalenol has also been used to reduce the virulence of Fusarium graminearum.
[0116] Genes used to help reduce Fusarium head blight include but are not limited to Tril 01 (Fusarium), PDR5 (yeast), tip-l(oat), tlp-2(oat), leaf tip- 1 (wheat), tip (rice), tlp-4 (oat), endochitinase, exochitinase, glucanase (Fusarium), permatin (oat), seed hordothionin (barley), alpha-thionin (wheat), acid glucanase (alfalfa), chitinase (barley and rice), class beta Π- 1,3 -glucanase (barley), PR5/tlp (Arabidopsis), zeamatin (maize), type 1 RTF (barley), NPR1 (Arabidopsis), lactoferrin (mammal), oxalyl-CoA-decarboxylase
(bacterium), LAP (baculovirus), ced-9 (C. elegans), and glucanase (rice and barley).
[0117] (B) A gene conferring resistance to a pest, such as Hessian fly, wheat stem soft fly, cereal leaf beetle, and/or green bug. For example the H9, H10, and H21 genes.
[0118] (C) A gene conferring resistance to such diseases as barley rusts, Septoria tritici, Septoria nodorum, powdery mildew, Helminthosporium diseases, smuts, bunts, Fusarium diseases, bacterial diseases, and viral diseases.
[0119] (D) A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications and hereby are incorporated by reference for this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO 91/14778; WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S. application Ser. Nos. 10/032,717; 10/414,637; and 10/606,320.
[0120] (E) An insect-specific hormone or pheromone such as an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344: 458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
[0121] (F) An insect-specific peptide that, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor); Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified in Diploptera puntata); Chattopadhyay et al. (2004) Critical Reviews in Microbiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2): 300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11):1515-1539; Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and
Vasconcelos & Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No. 5,266,317 to Tomalski et al, who disclose genes encoding insect-specific toxins.
[0122] (G) An enzyme responsible for a hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another nonprotein molecule with insecticidal activity.
[0123] (H) An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO
93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules that contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21 : 673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene, U.S. application Ser. Nos. 10/389,432,10/692,367, and U.S. Pat. No. 6,563,020.
[0124] (T) A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess et al., Plant Physiol. 104: 1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.
[0125] (J) A hydrophobic moment peptide. See PCT application WO 95/16776 and U.S. Pat. No. 5,580,852 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 and U.S. Pat. No. 5,607,914) (teaches synthetic antimicrobial peptides that confer disease resistance).
[0126] (K) A membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993), of heterologous expression of a cecropin-beta lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.
[0127] (L) A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev. Phytopathol. 28: 451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
[0128] (M) An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al, Abstract #497, Seventh International Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
[0129] (N) A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
[0130] (O) A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo-alpha-l,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha-l,4-D-galacturonase. See Lamb et al., Bio/Technology 10: 1436 (1992). The cloning and characterization of a gene which encodes a bean endo-poly-galacturonase-inhibiting protein is described by Toubart et al., Plant J. 2: 367 (1992).
[0131] (P) A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10: 305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
[0132] (Q) Genes involved in the Systemic Acquired Resistance (SAR) Response and/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2):128-131 (1995), Pieterse & Van Loon (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich (2003) Cell 11 3(7):815-6.
[0133] (R) Antifungal genes (Cornelissen and Melchers, PI. Physiol. 101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) and Bushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also see U.S. application Ser. No. 09/950,933.
[0134] (S) Detoxification genes, such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives. For example, see U.S. Pat. No. 5,792,931.
[0135] (T) Cystatin and cysteine proteinase inhibitors. See U.S. application Ser. No. 10/947,979.
[0136] (U) Defensin genes. See WO 03/000863 and U.S. application Ser. No. 10/178,213.
[0139] (V) Genes conferring resistance to nematodes. See WO 03/033651 and Urwin et. al., Planta 204:472-479 (1998), Williamson (1999) Curr. Opin. Plant Bio. 2(4):327-31.
2. Genes that Confer Resistance to an Herbicide, for Example:
[0137] (A) Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori et al. (1995) Mol Gen Genet 246:419). Other genes that confer tolerance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, and genes for various phosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619).
[0138] (B) An herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and ABAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449 (1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270, which are incorporated herein by reference for this purpose.
[0139] (C) Glyphosate (resistance imparted by mutant 5-enolpyruvl-3-phosphoshikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus PAT (bar) genes), and pyridinoxy or phenoxy proprionic acids and cycloshexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses the nucleotide sequence of a form of EPSPS that can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes. See also U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 Bl; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 Bl; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;
5,510,471; Re. 36,449; RE 37,287 E; and U.S. Pat. No. 5,491,288; and international publications EP1173580; WO 01/66704; EP1173581 and EP1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene that encodes a glyphosate oxido-reductase enzyme as described more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition glyphosate resistance can be imparted to plants by the over-expression of genes encoding glyphosate N-acetyltransferase. See, for example, U.S. application Ser. Nos. 10/46227, 10/427,692 and 10/427,692. ADNA molecule encoding a mutant aroA gene can be obtained under ATCC accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a PAT gene is provided in European Patent No. 0242 246 and 0 242236 to Leemans et al. De Greef et al., Bio/Technology 7: 61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for PAT activity. See also, U.S. Pat. Nos.
5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 Bl; and U.S. Pat. No. 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cycloshexones, such as sethoxydim and haloxyfop, are the Accl-Sl, Accl-S2 and Accl- S3 genes described by Marshall et al, Theor. Appl. Genet. 83: 435 (1992).
[0140] (D) An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al, Biochem. J.285:173 (1992).
[0141] (E) Protoporphyrinogen oxidase (protox) is necessary for the production of chlorophyll, which is necessary for all plant survival. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Pat. Nos. 6,288,306 Bl; 6,282,837 Bl; and 5,767,373; and international publication WO 01/12825.
3. Genes that Confer or Improve Grain Quality, such as:
[0142] (A) Altered fatty acids, for example, by (1) down-regulation of stearyl-ACP desaturase to increase stearic acid content of the plant, by for example, transforming a plant with a nucleic acid encoding an anti-sense of stearyl-ACP desaturase. See Knultzon et al, Proc. Natl. Acad. Sci. USA 89: 2624 (1992) and W099/64579 (Genes for
Desaturases to Alter Lipid Profiles in Corn), (2) Elevating oleic acid via FAD-2 gene modification and/or decreasing linolenic acid viaFAD-3 gene modification (see U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245), [0082] (3) Altering conjugated linolenic or linoleic acid content, such as in WO 01/12800, [0083] (4) Altering LEC1, AGP, Dekl, Superall, milps, and various lpa genes such as lpal, lpa3, hpt or hggt. For example, see WO 02/42424, WO 98/22604, WO 03/011015, U.S. Pat. No. 6,423,886, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,825,397, US 2003/0079247, US 2003/0204870, WO 02/057439, WO 03/011015 and Rivera-Madrid, R. et al. Proc. Natl. Acad. Sci. 92:5620- 5624 (1995).
[0143] (B) Altered phosphorus content, for example, the (1) Introduction of aphytase- encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127: 87 (1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene. (2) Up- regulation of a gene that reduces phytate content. In maize for example, this could be accomplished by cloning and then re-introducing DNA associated with one or more of the alleles, such as the LPA alleles, identified in maize mutants characterized by low levels of phytic acid, such as in Raboy et al., Maydica 35: 383 (1990) and/or by altering inositol kinase activity as in WO 02/059324, US 2003/0009011, WO 03/027243, US
2003/0079247, WO 99/05298, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, WO 2002/059324, US 2003/0079247, WO 98/45448, WO 99/55882, WO 01/04147.
[0144] (C) Altered carbohydrates effected, for example, by altering a gene for an enzyme that affects the branching pattern of starch or a gene altering thioredoxin (See U.S. Pat. No. 6,531,648). See Shiroza et al, J. Bacteriol. 170: 810 (1988) (nucleotide sequence of Streptococcus mutans fructosyltransferase gene), Steinmetz et al, Mol. Gen. Genet. 200: 220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase.gene), Pen et al., Bio/Technology 10: 292 (1992) (production of transgenic plants that express Bacillus licheniformis alpha-amylase), Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes), Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis of barley alpha-amylase gene), and Fisher et al., Plant Physiol. 102: 1045 (1993) (maize endosperm starch branching enzyme Π), WO 99/10498
(improved digestibility and/or starch extraction through modification of UDP-D-xylose 4- epimerase, Fragile 1 and 2, Refl, HCHL, C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed by modification of starch levels (AGP)). The fatty acid modification genes mentioned above may also be used to affect starch content and/or composition through the interrelationship of the starch and oil pathways.
[0145] (D) Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683, US2004/0034886 and WO
00/68393 involving the manipulation of antioxidant levels through alteration of a phytl prenyl transferase (ppt), WO 03/082899 through alteration of a homogentisate geranyl geranyl transferase (hggt). [0146] (E) Altered essential seed amino acids. For example, see U.S. Pat. No. 6,127,600 (method of increasing accumulation of essential amino acids in seeds), U.S. Pat. No. 6,080,913 (binary methods of increasing accumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), WO 99/40209 (alteration of amino acid compositions in seeds), WO 99/29882 (methods for altering amino acid content of proteins), U.S. Pat. No. 5,850,016 (alteration of amino acid compositions in seeds), WO 98/20133 (proteins with enhanced levels of essential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plant amino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increased lysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophan synthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolic enzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414 (increased memionine), WO 98/56935 (plant amino acid biosynthetic enzymes), WO 98/45458 (engineered seed protein having higher percentage of essential amino acids), WO 98/42831 (increased lysine), U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S. Pat. No. 5,559,223 (synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants), WO 96/01905 (increased threonine), WO 95/15392 (increased lysine), US 2003/0163838, US 2003/0150014, US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516, and WO 00/09706 (Ces A: cellulose synthase), U.S. Pat. No. 6,194,638 (hemicellulose), U.S. Pat. No. 6,399,859 and US 2004/0025203 (UDPGdH), U.S. Pat. No. 6,194,638 (RGP).
4. Genes that Control Male-Sterility
[0147] There are several methods of conferring genetic male sterility available, such as multiple mutant genes at separate locations within the genome that confer male sterility, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations as described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. In addition to these methods, Albertsen et al., U.S. Pat. No. No. 5,432,068, describe a system of nuclear male sterility which includes: identifying a gene which is critical to male fertility; silencing this native gene which is critical to male fertility; removing the native promoter from the essential male fertility gene and replacing it with an inducible promoter; inserting this genetically engineered gene back into the plant; and thus creating a plant that is male sterile because the inducible promoter is not "on" resulting in the male fertility gene not being transcribed. Fertility is restored by inducing, or turning "on", the promoter, which in turn allows the gene that confers male fertility to be transcribed.
[0148] (A) Introduction of a deacetylase gene under the control of a tapetum-specific promoter and with the application of the chemical N-Ac-PPT (WO 01/29237).
[0149] (B) Introduction of various stamen-specific promoters (WO 92/13956, WO 92/13957).
[0150] (C) Introduction of the barnase and the barstar gene (Paul et al. Plant Mol. Biol. 19:611-622, 1992).
[0151] For additional examples of nuclear male and female sterility systems and genes, see also, U.S. Pat. No. 5,859,341; U.S. Pat. No. 6,297,426; U.S. Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S. Pat. No. 5,850,014; and U.S. Pat. No. 6,265,640; all of which are hereby incorporated by reference.
5. Genes that Create a Site for Site Specific DNA Integration.
[0152] This includes the introduction of FRT sites that may be used in the FLP/ERT system and/or Lox sites that may be used in the Cre/Loxp system. For example, see
Lyznik, et al., Site-Specific Recombination for Genetic Engineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821, which are hereby incorporated by reference. Other systems that may be used include the Gin recombinase of phage Mu (Maeser et al., 1991; Vicki Chandler, The Maize Handbook chapter 118 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto et al., 1983), and the R/RS system of the pSRl plasmid (Araki et al., 1992).
6. Genes that Affect Abiotic Stress Resistance
[0153] (A) These may include but are not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance and increased yield under stress. For example, see: WO 00/73475 where water use efficiency is altered through alteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat. No. 6,717,034, U.S. Pat. No. 6,801,104, WO2000060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO
2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO 2003/013227, WO
2003/013228, WO 2003/014327, WO 2004/031349, WO 2004/076638, WO 98/09521, and WO 99/38977 describing genes, including CBF genes and transcription factors effective in mitigating the negative effects of freezing, high salinity, and drought on plants, as well as conferring other positive effects on plant phenotype; US 2004/0148654 and WO 01/36596 where abscisic acid is altered in plants resulting in improved plant phenotype such as increased yield and/or increased tolerance to abiotic stress; WO 2000/006341, WO 04/090143, U.S. application Ser. Nos. 10/817483 and 09/545,334 where cytolcinin expression is modified resulting in plants with increased stress tolerance, such as drought tolerance, and/or increased yield. Also see WO 02/02776, WO
2003/052063, JP2002281975, U.S. Pat. No. 6,084,153, WO 01/64898, U.S. Pat. No. 6,177,275 and U.S. Pat. No. 6,107,547 (enhancement of nitrogen utilization and altered nitrogen responsiveness). For ethylene alteration, see US 20040128719, US 20030166197 and WO 2000/32761. For plant transcription factors or transcriptional regulators of abiotic stress, see e.g. US 2004/0098764 or US 2004/0078852.
[0154] (B) Improved tolerance to water stress from drought or high salt water condition. The HVA1 protein belongs to the group 3 LEA proteins that include other members such as wheat pMA2005 (Curry et al, 1991; Curry and Walker-Simmons, 1993), cotton D-7 (Baker et al., 1988), carrot Dc3 (Seffens et al., 1990), and rape pLEA76 (Harada et al., 1989). These proteins are characterized by 11-mer tandem repeats of amino acid domains which may form a probable amphophilic alpha-helical structure that presents a hydrophilic surface with a hydrophobic stripe (Baker et al., 1988; Dure et al, 1988; Dure, 1993). The barley HVA1 gene and the wheat p A2005 gene (Curry et al., 1991; Curry and Walker- Simmons, 1993) are highly similar at both the nucleotide level and predicted amino acid level. These two monocot genes are closely related to the cotton D-7 gene (Baker et al., 1988) and carrot Dc3 gene (Seffens et al., 1990) with which they share a similar structural gene organization (Straub et al., 1994). There is, therefore, a correlation between LEA gene expression or LEA protein accumulation with stress tolerance in a number of plants. For example, in severely dehydrated wheat seedlings, the accumulation of high levels of group 3 LEA proteins was correlated with tissue dehydration tolerance ( ied and Walker- Simmons, 1993). Studies on several Indica varieties of rice showed that the levels of group 2 LEA proteins (also known as dehydrins) and group 3 LEA proteins in roots were significantly higher in salt-tolerant varieties compared with sensitive varieties (Moons et al, 1995). The barley HVA1 gene was transformed into wheat. Transformed wheat plants showed increased tolerance to water stress, (Sivamani, E. et al. Plant Science (2000), V. 155 pl-9 and U.S. Pat. No. 5,981,842.) [0155] (C) Improved water stress tolerance through increased mannitol levels via the bacterial mannitol- 1 -phosphate dehydrogenase gene. To produce a plant with a genetic basis for coping with water deficit, Tarczynski et al. (Proc. Natl. Acad. Sci. USA, 89, 2600 (1992); WO 92/19731, published No. 12, 1992; Science, 259, 508 (1993)) introduced the bacterial mannitol- 1 -phosphate dehydrogenase gene, mtID, into tobacco cells via
Agrobacterium-mediated transformation. Root and leaf tissues from transgenic plants regenerated from these transformed tobacco cells contained up to 100 mM mannitol. Control plants contained no detectable mannitol. To determine whether the transgenic tobacco plants exhibited increased tolerance to water deficit, Tarczynski et al. compared the growth of transgenic plants to that of untransformed control plants in the presence of 250 mM NaCl. After 30 days of exposure to 250 mM NaCl, transgenic plants had decreased weight loss and increased height relative to their untransformed counterparts. The authors concluded that the presence of mannitol in these transformed tobacco plants contributed to water deficit tolerance at the cellular level. See also U.S. Pat. No. 5,780,709 and international publication WO 92/19731 which are incorporated herein by reference for this purpose.
[0156] Other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and/or plant structure, can be introduced or introgressed into plants, see e.g. WO 97/49811 (LHY), W098/56918 (ESD4),
WO97/10339 and U.S. Pat. No. 6,573,430 (TEL), U.S. Pat. No. 6,713,663 (FT), WO 96/14414 (CON), WO 96/38560, WO01/21822 (VRN1), WO 00/44918 (VRN2), WO 99/49064 (GI), WO 00/46358 (FBI), WO 97/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No. 6,307,126 (GAT), WO 99/09174 (D8 and R t), and WO2004076638 and WO2004031349 (transcription factors).
7. Genes that Confer Agronomic Enhancements, Nutritional Enhancements, or Industrial Enhancements.
[0157] Altered enzyme activity for improved disease resistance and/or improved plant or grain quality. For example lipoxygenase levels can be altered to improve disease resistance (Steiner-Lange, S., et al. 2003. MPMI. 16(10):893-902. Differential defense reactions in leaf tissues of barley in response to infection by Rh nchosporium secalis and to treatment with a fungal avirulence gene product) and/or to improve the quality of the grain resulting in improved flavor for beer, cereal and other food products made from the grain (Douma, A., et al. 2003. U.S. Pat. No. 6,660,915). Another enzyme whose activity can be altered is beta-glucanase for improved plant and/or grain quality (Han, F., et al. 1995. Mapping of beta-glucan content and beta-glucanase activity loci in barley grain and malt. Theor. Appl. Genet. 91:921-927; Han, F., et al. 1997. Towards fine structure mapping and tagging major malting quality QTL in barley. Theor. Appl. Genet. 95:903- 910; Jensen, L. G., et al. 1996. Transgenic barley expressing a protein-engineered, thermostable (l,3-l,4)-beta-glucanase during germination. Proc. Natl. Acad. Sci. U.S.A. 93(8):3487-3491). Yet another enzyme whose activity can be altered is polyphenol oxidase for improved plant and/or grain quality (Cahoon, R. 2004. U.S. Patent Publication 2004/0214201).
Mutation Breeding
[0158] Mutation breeding is another method of introducing new traits into barley variety MV115. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g. cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900 nrn), or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques. Details of mutation breeding can be found in
"Principles of Cultivar Development" Fehr, 1993 Macmillan Publishing Company. In addition, mutations created in other barley plants may be used to produce a backcross conversion of barley cultivar MV115 that comprises such mutation. Backcross Conversion of MV115
[0159] A further embodiment of the invention is a backcross conversion of barley variety MV11 . A backcross conversion occurs when DNA sequences are introduced through traditional (non-transformation) breeding techniques, such as backcrossing. DNA sequences, whether naturally occurring or transgenes, may be introduced using these traditional breeding techniques. Desired traits transferred through this process include, but are not limited to nutritional enhancements, industrial enhancements, disease resistance, insect resistance, herbicide resistance, agronomic enhancements, grain quality
enhancement, waxy starch, breeding enhancements, seed production enhancements, and male sterility. Descriptions of some of the cytoplasmic male sterility genes, nuclear male sterility genes, chemical hybridizing agents, male fertility restoration genes, and methods of using the aforementioned are discussed in Hybrid Wheat by K. A. Lucken (pp. 444-452 In Wheat and Wheat Improvement, ed. Heyne, 1987). Examples of genes for other traits include: Leaf rust resistance genes (Lr series such as Lrl, LrlO, Lr21, Lr22, Lr22a, Lr32, Lr37, Lr41, Lr42, and Lr43), Fusarium head blight-resistance genes (QFhs.ndsu-3B and QFhs.ndsu-2A), Powdery Mildew resistance genes (Pm21), common bunt resistance genes (Bt-10), and wheat streak mosaic virus resistance gene (Wsml), Russian wheat aphid resistance genes (Dn series such as Dnl, Dn2, Dn4, Dn5), Black stem rust resistance genes (Sr38), Yellow rust resistance genes (Yr series such as Yrl, YrSD, Yrsu, Yrl7, Yrl5, YrH52), Aluminum tolerance genes (Alt(BH)), dwarf genes (Rht), vernalization genes (Vrn), Hessian fly resistance genes (H9, H10, H21, H29), grain color genes (R/r), glyphosate resistance genes (EPSPS), glufosinate genes (bar, pat) and water stress tolerance genes (Hval, mtID). The trait of interest is transferred from the donor parent to the recurrent parent, in this case, the barley plant disclosed herein. Single gene traits may result from either the transfer of a dominant allele or a recessive allele. Selection of progeny containing the trait of interest is done by direct selection for a trait associated with a dominant allele. Selection of progeny for a trait that is transferred via a recessive allele requires growing and selfing the first backcross to determine which plants carry the recessive alleles. Recessive traits may require additional progeny testing in successive backcross generations to determine the presence of the gene of interest.
[0160] Another embodiment of this invention is a method of developing a backcross conversion MVl 15 barley plant that involves the repeated backcrossing to barley variety MV115. The number of backcrosses made may be 2, 3, 4, 5, 6 or greater, and the specific number of backcrosses used will depend upon the genetics of the donor parent and whether molecular markers are utilized in the backcrossing program. See, for example, von Bothmer, R. et al. 2003. Diversity in Barley (Elsevier Science) and Slafer, G. et al. 2002. Barley Science: Recent Advances from Molecular Biology to Agronomy of Yield and Quality (Haworth Press). Using backcrossing methods, one of ordinary skill in the art can develop individual plants and populations of plants that retain at least 70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the genetic profile of barley variety MV115. The percentage of the genetics retained in the backcross conversion may be measured by either pedigree analysis or through the use of genetic techniques such as molecular markers or electrophoresis. In pedigree analysis, on average 50% of the starting germplasm would be passed to the progeny line after one cross to another line, 75% after backcrossing once, 87.5% after backcrossing twice, and so on. Molecular markers could also be used to confirm and/or determine the recurrent parent used. The backcross conversion developed from this method may be similar to MV115 for the results listed in Table 1. Such similarity may be measured by a side by side phenotypic comparison, with differences and similarities determined at a 5% significance level. Any such comparison should be made in environmental conditions that account for the trait being transferred. For example, herbicide should not be applied in the phenotypic comparison of herbicide resistant backcross conversion of MV115 to MV115.
[0161] Another embodiment of the invention is an essentially derived variety of MV115. As determined by the UPOV Convention, essentially derived varieties may be obtained for example by the selection of a natural or induced mutant, or of a somaclonal variant, the selection of a variant individual from plants of the initial variety, backcrossing, or transformation by genetic engineering. An essentially derived variety of MV115 is further defined as one whose production requires the repeated use of variety MV115 or is predominately derived from variety MV115. International Convention for the Protection of New Varieties of Plants, as amended on Mar. 19, 1991, Chapter V, Article 14, Section 5(c).
[0162] This invention also is directed to methods for using barley variety MV115 in plant breeding. One such embodiment is the method of crossing barley variety MV115 with another variety of barley to form a first generation population of Fi plants. The population of first generation Fi plants produced by this method is also an embodiment of the invention. This first generation population of Fi plants will comprise an essentially complete set of the alleles of barley variety MV115. One of ordinary skill in the art can utilize either breeder books or molecular methods to identify a particular Fi plant produced using barley variety MV115, and any such individual plant is also encompassed by this invention. These embodiments also cover use of transgenic or backcross conversions of barley variety MV115 to produce first generation Fi plants.
[0163] A method of developing a MV115-progeny barley plant comprising crossing MVl 15 with a second barley plant and performing a breeding method is also an embodiment of the invention. A specific method for producing a line derived from barley variety MVl 15 is as follows. One of ordinary skill in the art would cross barley variety MVl 15 with another variety of barley, such as an elite variety. The Fi seed derived from this cross would be grown to form a homogeneous population. The Fi seed would contain one set of the alleles from variety MVl 15 and one set of the alleles from the other barley variety. The Fi genome would be made-up of 50% variety MVl 15 and 50% of the other elite variety. The Fi seed would be grown and allowed to self, thereby forming F2 seed. On average the F2 seed would have derived 50% of its alleles from variety MVl 15 and 50% from the other barley variety, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15 (Wang J. and R. Bernardo, 2000, Crop Sci. 40:659-665 and Bernardo, R. and A. L. Kahler, 2001, Theor. Appl. Genet 102:986-992). The F2 seed would be grown and selection of plants would be made based on visual observation and/or measurement of traits. The MVl 15 -derived progeny that exhibit one or more of the desired MVl 15-derived traits would be selected and each plant would be harvested separately. This F3 seed from each plant would be grown in individual rows and allowed to self. Then selected rows or plants from the rows would be harvested and threshed individually. The selections would again be based on visual observation and/or measurements for desirable traits of the plants, such as one or more of the desirable MVl 15-derived traits. The process of growing and selection would be repeated any number of times until a homozygous MVl 15-derived barley plant is obtained. The homozygous MVl 15-derived barley plant would contain desirable traits derived from barley variety MVl 15, some of which may not have been expressed by the other original barley variety to which barley variety MVl 15 was crossed and some of which may have been expressed by both barley varieties but now would be at a level equal to or greater than the level expressed in barley variety MVl 15. The homozygous MVl 15- derived barley plants would have, on average, 50% of their genes derived from barley variety MVl 15, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15. The breeding process, of crossing, selfing, and selection may be repeated to produce another population of MVl 15-derived barley plants with, on average, 25% of their genes derived from barley variety MVl 15, but various individual plants from the population would have a much greater percentage of their alleles derived from MVl 15. Another embodiment of the invention is a homozygous MVl 15-derived barley plant that has received MVl 15-derived traits. [0164] The previous example can be modified in numerous ways, for instance selection may or may not occur at every selfing generation, selection may occur before or after the actual self-pollination process occurs, or individual selections may be made by harvesting individual spikes, plants, rows or plots at any point during the breeding process described. In addition, double haploid breeding methods may be used at any step in the process. The population of plants produced at each and any generation of selfmg is also an embodiment of the invention, and each such population would consist of plants containing
approximately 50% of its genes from barley variety MV115, 25% of its genes from barley variety MV115 in the second cycle of crossing, selfing, and selection, 12.5% of its genes from barley variety MV115 in the third cycle of crossing, selfing, and selection, and so on.
[0165] Another embodiment of this invention is the method of obtaining a homozygous MV115-derived barley plant by crossing barley variety MV115 with another variety of barley and applying double haploid methods to the Fi seed or F] plant or to any generation of MV115-derived barley obtained by the selfing of this cross.
[0166] Still further, this invention also is directed to methods for producing MV115- derived barley plants by crossing barley variety MV115 with a barley plant and growing the progeny seed, and repeating the crossing or selfing along with the growing steps with the MV115-derived barley plant from 1 to 2 times, 1 to 3 times, 1 to 4 times, or 1 to .5 times. Thus, any and all methods using barley variety MVT15 in breeding are part of this invention, including selfing, pedigree breeding, backcrossing, hybrid production and crosses to populations. Unique starch profiles, molecular marker profiles and/or breeding records can be used by those of ordinary skill in the art to identify the progeny lines or populations derived from these breeding methods.
[0167] In addition, this invention also encompasses progeny with the same or greater yield or test weight of MV115, the same or shorter plant height, and the same or greater resistance to smut, stem rust, Septoria, net and spot blotch of MV115. The expression of these traits may be measured by a side by side phenotypic comparison, with differences and similarities determined at a 5% significance level. Any such comparison should be made in the same environmental conditions.
[0168] In one aspect of the present invention, barley cultivar MV115, was tested for malt protein production when grown in plots in one environment in Idaho in 2008, 2009 and 2010. Comparisons between MV115 and two Moravian cultivars, Moravian 037 and Moravian 069, are shown in Table 2 (2008 data) Table 3 (2009 data), Table 4 (2010 data). Table 2
% Beta-
2008 % Test Plant Extract Glucan FAN AA DP bu/ % Protein Weight Height % Maturity Dry Dry Dry Diy Dry Malt
Year Variety acre Plump (NIR) (lbs) (in) Lodging (HIB) Basis Basis Basis Basis Basis Viscosity Protein
2008 Moravian 037 230 98.4 12.7 56.7 33 0.0 79.2 435 127 40 104 1.984 12.7
2008 Moravian 069 250 96.3 11.8 55.3 31 3.3 81.2 349 132 54 114 1.593 10.9
2008 Moravian 115 238 98.5 12.4 55.3 30 3.3 79.3 297 132 53 107 1.587 11.1
Trial Mean 233 97.4 12.3 54.9 32 2.9 80.6 401 137 58 115 1.630 11.8
PLSD
(p=0.05) 21.0 1.47 0.91 0.85 2 14.2
Table 3
% Beta-
2009 % Test Plant Extract Glucan FAN AA DP bu/ % Protein Weight Height % Maturity Dry Dry Dry Dry Dry Malt
Year Variety acre Plump (NIR) (lbs) (in) Lodging (HIB) Basis Basis Basis Basis Basis Viscosity Protein
2009 Moravian 037 219 97.6 11.8 57.4 30.7 0.0 2.3 79.4 615 94 44 97 2.019 11.5
2009 Moravian 069 199 95.2 10.9 55.7 29.7 7.3 2.0 82.5 287 165 63 103 1.597 10.1
2009 Moravian 115 194 93.3 12.4 55.1 32.3 10.0 1.0 80.6 97 188 68 112 1.485 11.1
Trial Mean 210 93.3 12.2 55.4 32.8 16.9 81.6 345 167 71 123 1.622 11.4
PLSD
(p=0.05) 50 10.4 3.1 2.3 5.5 56.7
Table 4
% Beta-
% Test Plant Extract Glucan FAN AA DP
2010 % Protein Weight Height % Maturity Dry Diy Dry Dry Dry Malt
Year Variety Bu/acre Plump (NIR) (lbs) . (in) Lodging (HIB) Basis Basis Basis Basis Basis Viscosity Protein
2010 Moravian 037 167 98.2 10.8 57.0 36.0 0.0 2.3 80.1 368 118 47 109 1.7548 10.5
2010 Moravian 069 179 96.4 10.0 56.0 32.5 0.0 2.3 81.0 218 166 74 111 1.5167 9.6
2010 Moravian 115 188 97.4 9.4 54.8 30.7 0.0 2.0 84.1 110 174 77 107 1.5458 9.0
Trial Mean 180 80.8 8.4 46.1 28.0 0.1 1.8 82.3 219 173 68 104 1.5514 9.6
PLSD
(p=0.05) 50.4 2.8 2 1.3 4.4 19.4 1.4
[0169] In Tables 2-4, column one shows the year, column two shows the variety, column three shows the bushels per acre (bu/care), column four shows the percent plump (% plump), column five shows the % protein content (% protein NTR), column six shows the test weight in pounds, column seven shows the plant height in inches, column eight shows the percent lodging, column nine shows the maturity HOB, column ten shows the percent extract on a dry basis, column eleven shows the beta-glucan content on a dry basis, column twelve shows the FAN on a dry basis, column thirteen shows the AA on a dry- basis, column fourteen shows the DP on a dry basis, column fifteen shows the viscosity, and, column sixteen shows the mal protein content.
DEPOSIT INFORMATION
[0170] A deposit of the MillerCoors proprietary barley cultivar designated MV115 disclosed above and recited in the appended claims will be made under the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Upon allowance of any claims i this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to the deposit with the American Type Culture Collection, Manassas, Va. The deposit will be maintained in the depository for a period of 30. years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
[0171] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter are interpreted to include all such modifications, permutations, additions, and sub-combinations as are within their true spirit and scope.

Claims

What is claimed
1. A seed of barley cultivar Moravian 115 (MV115).
2. A barley plant, or a part thereof, produced by growing the seed of claim 1.
3. A tissue culture produced from protoplasts or cells from the plant of claim 2, wherein said protoplasts or cells are produced from a plant part selected from the group consisting of head, awn, leaf, pollen, embryo, cotyledon, hypocotyl, seed, spike, pericarp, meristematic cell, root, root tip, pistil, anther, floret, shoot, stem and callus.
4. A barley plant regenerated from the tissue culture of claim 3, wherein the plant has all of the morphological and physiological characteristics of barley cultivar MV 115.
5. A method for producing a barley seed, wherein the method comprises crossing two barley plants and harvesting the resultant barley seed, wherein at least one barley plant is the barley plant of claim 2.
6. A barley seed produced by the method of claim 5.
7. A barley plant, or a part thereof, produced by growing said seed of claim 6.
8. The method of claim 5, wherein at least one of said barley plants is transgenic.
9. A method of producing an herbicide resistant barley plant, wherein said method comprises introducing a gene conferring herbicide resistance into the plant of claim 2.
10. An herbicide resistant barley plant produced by the method of claim 9, the gene confers resistance to an herbicide selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L- phosphinothricin, cyclohexone, cyclohexanedione, triazine, and benzonitrile.
11. A method of producing a pest or insect resistant barley plant, wherein the method comprises introducing a gene conferring pest or insect resistance into the barley plant of claim 2.
12. A pest or insect resistant barley plant produced by the method of claim 11.
13. The barley plant of claim 12, wherein the gene encodes a Bacillus thuringiensis (Bt) endotoxin.
14. A method of producing a disease resistant barley plant, wherein the method comprises introducing a gene conferring disease resistance into the barley plant of claim 2.
15. A disease resistant barley plant produced by the method of claim 14.
16. A method of producing a barley plant with modified fatty acid metabolism, modified carbohydrate metabolism or modified protein metabolism, wherein the method comprises introducing a gene encoding a protein selected from the group consisting of modified glutenins, gliadins, phytase, lipoxygenase, beta-glucanase, polyphenol oxidase, fructosyltransferase, levansucrase, a-amylase, invertase and starch branching enzyme or encoding an antisense of stearyl-ACP desaturase into the barley plant of claim 2.
17. A barley plant having modified fatty acid metabolism, modified carbohydrate metabolism or modified protein metabolism produced by the method of claim 16.
18. A method of introducing a desired trait into barley cultivar MVl 15, wherein the method comprises:(a) crossing a MVl 15 plant, with a plant of another barley cultivar that comprises a desired trait to produce progeny plants wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, modified fatty acid metabolism, modified carbohydrate metabolism, modified phytic acid metabolism, modified waxy starch content, modified protein content, increased stress to water tolerance and resistance to bacterial disease, fungal disease or viral disease;(b) selecting one or more progeny plants that have the desired trait to produce selected progeny plants;(c) crossing the selected progeny plants with the MVl 15 plants to produce backcross progeny plants;(d) selecting for backcross progeny plants that have the desired trait and all of the physiological and morphological characteristics of barley cultivar MVl 15 listed in Table 1; and(e) repeating steps (c) and (d) two or more times in succession to produce selected third or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of barley cultivar M 115 listed in Table 1.
19. A barley plant produced by the method of claim 18, wherein the plant has the desired trait.
20. The barley plant of claim 19, wherein the desired trait is herbicide resistance and the resistance conferred is to an herbicide selected from the group consisting of
imidazolinone, dicamba, cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxy proprionic acid, L-phosphinothricin, triazine and benzonitrile.
21. The barley plant of claim 19, wherein the desired trait is insect resistance and the insect resistance is conferred by a gene encoding a Bacillus thuringiensis endotoxin.
22. The barley plant of claim 19, wherein the desired trait is modified fatty acid metabolism, modified carbohydrate metabolism or modified protein metabolism and said desired trait is conferred by a nucleic acid encoding a protein selected from the group consisting of modified glutenins, gliadins, phytase, lipoxygenase, beta-glucanase, polyphenol oxidase, fructosyltransferase, levansucrase, a-amylase, invertase and starch branching enzyme or encoding an antisense of stearyl-ACP desaturase.
23. The barley plant of claim 19, wherein the desired trait is male sterility and the trait is conferred by a nucleic acid molecule that confers male sterility.
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