Low saturated-fat sunflower and associated methods
10045503 ยท 2018-08-14
Assignee
Inventors
- James Todd Gerdes (Breckenridge, MN, US)
- Charles James Kahl (Westfield, IN, US)
- Angela Lee Erickson (Fergus Falls, MN, US)
- Robert Martin Benson (Ellsworth, WI, US)
Cpc classification
A01H1/02
HUMAN NECESSITIES
C12N15/82
CHEMISTRY; METALLURGY
A23V2002/00
HUMAN NECESSITIES
International classification
A01H5/00
HUMAN NECESSITIES
C12N15/82
CHEMISTRY; METALLURGY
Abstract
Provided are sunflowers, parts thereof, cultures of, and seeds that are capable of producing sunflower oil that is low in saturated fat as well as associated methods.
Claims
1. A method of producing a sunflower plant comprising a seed oil trait and a desired trait selected from the group consisting of male sterility, herbicide resistance, insect resistance, and resistance to bacterial disease, fungal disease, or viral disease, the method comprising: crossing a sunflower plant of a first sunflower cultivar with a sunflower plant of a second sunflower cultivar to produce F.sub.1 progeny plants, wherein the first sunflower cultivar produces seed having a seed oil trait selected from the group consisting of (i) a seed oil fatty acid content comprising 10% or less total combined palmitic acid and stearic acid content and 15% or more linoleic acid content, and (ii) a seed oil fatty acid content comprising 3.3% or less total combined palmitic acid and stearic acid content, wherein the genome of the first sunflower cultivar comprises the polynucleotide of SEQ ID NO:13, comprising a cytosine at position 60 of the polynucleotide, a guanine at position 181 of the polynucleotide, a cytosine at position 258 of the polynucleotide, and a thymine at position 261 of the polynucleotide, and wherein the second sunflower cultivar comprises a desired trait selected from the group consisting of male sterility, herbicide resistance, insect resistance, and resistance to bacterial disease, fungal disease, or viral disease; and selecting a progeny plant that has the desired trait and produces seed having the seed oil trait, wherein the selected progeny plant and the seed oil derived thereof comprises in its genome the polynucleotide of SEQ ID NO: 13, comprising a cytosine at position 60 of the polynucleotide, a guanine at position 181 of the polynucleotide, a cytosine at position 258 of the polynucleotide, and a thymine at position 261 of the polynucleotide, thereby producing a selected progeny plant comprising the seed oil trait and the desired trait.
2. The method according to claim 1, wherein the seed oil trait is a seed oil fatty acid profile comprising 10% or less total combined palmitic acid and stearic acid content and 15% or more linoleic acid content.
3. The method according to claim 1, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.3% or less total combined palmitic acid and stearic acid content.
4. The method according to claim 3, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.0% or less total combined palmitic acid and stearic acid content.
5. The method according to claim 1, wherein the genome of the first sunflower cultivar comprises the polynucleotide of SEQ ID NO: 18, comprising an adenine at position 299 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 364 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 439 of the polynucleotide of SEQ ID NO: 18, and an adenine at position 457 of the polynucleotide of SEQ ID NO: 18, and wherein the selected progeny plant and seed oil derived thereof comprises in its genome the polynucleotide of SEQ ID NO: 18, comprising an adenine at position 299 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 364 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 439 of the polynucleotide of SEQ ID NO: 18, and an adenine at position 457 of the polynucleotide of SEQ ID NO:18.
6. The method according to claim 5, wherein the seed oil trait is a seed oil fatty acid profile comprising 10% or less total combined palmitic acid and stearic acid content and 15% or more linoleic acid content.
7. The method according to claim 5, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.3% or less total combined palmitic acid and stearic acid content.
8. The method according to claim 7, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.0% or less total combined palmitic acid and stearic acid content.
9. The method according to claim 1, the method further comprising: backcrossing said selected progeny plant with a sunflower plant of the first sunflower cultivar to produce backcross progeny plants; selecting at least one backcross progeny plant that has the desired trait and produces seed having the seed oil trait, wherein the selected backcross progeny plant and the seed oil derived thereof comprises in its genome the polynucleotide of SEQ ID NO: 13, comprising a cytosine at position 60 of the polynucleotide, a guanine at position 181 of the polynucleotide, a cytosine at position 258 of the polynucleotide, and a thymine at position 261 of the polynucleotide; and repeating the backcrossing step and the backcrossing selection step three or more times, thereby producing a selected fourth or higher backcross progeny plant comprising the seed oil trait and the desired trait.
10. The method according to claim 9, wherein the seed oil trait is a seed oil fatty acid profile comprising 10% or less total combined palmitic acid and stearic acid content and 15% or more linoleic acid content.
11. The method according to claim 9, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.3% or less total combined palmitic acid and stearic acid content.
12. The method according to claim 11, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.0% or less total combined palmitic acid and stearic acid content.
13. The method according to claim 9, wherein the genome of the first sunflower cultivar comprises the polynucleotide of SEQ ID NO: 18, comprising an adenine at position 299 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 364 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 439 of the polynucleotide of SEQ ID NO: 18, and an adenine at position 457 of the polynucleotide of SEQ ID NO: 18, wherein the selected progeny plant and the seed oil derived thereof comprises in its genome the polynucleotide of SEQ ID NO: 18, comprising an adenine at position 299 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 364 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 439 of the polynucleotide of SEQ ID NO: 18, and an adenine at position 457 of the polynucleotide of SEQ ID NO: 18, and wherein the selected fourth or higher backcross progeny plant and the seed oil derived thereof comprises in its genome the polynucleotide of SEQ ID NO: 18, comprising an adenine at position 299 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 364 of the polynucleotide of SEQ ID NO: 18, a cytosine at position 439 of the polynucleotide of SEQ ID NO: 18, and an adenine at position 457 of the polynucleotide of SEQ ID NO: 18.
14. The method according to claim 13, wherein the seed oil trait is a seed oil fatty acid profile comprising 10% or less total combined palmitic acid and stearic acid content and 15% or more linoleic acid content.
15. The method according to claim 13, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.3% or less total combined palmitic acid and stearic acid content.
16. The method according to claim 15, wherein the seed oil trait is a seed oil fatty acid profile comprising 3.0% or less total combined palmitic acid and stearic acid content.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
(4) In the description and tables that follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:
(5) Allele. Allele is any of one or more alternative forms of a gene, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
(6) Backcrossing. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F.sub.1 with one of the parental genotypes of the F.sub.1 hybrid.
(7) Elite sunflower. A sunflower cultivar which has been stabilized for certain commercially important agronomic traits comprising a stabilized yield of about 100% or greater relative to the yield of check varieties in the same growing location growing at the same time and under the same conditions. In one embodiment, elite sunflower means a sunflower cultivar stabilized for certain commercially important agronomic traits comprising a stabilized yield of 110% or greater relative to the yield of check varieties in the same growing location growing at the same time and under the same conditions. In another embodiment, elite sunflower means a sunflower cultivar stabilized for certain commercially important agronomic traits comprising a stabilized yield of 115% or greater relative to the yield of check varieties in the same growing location growing at the same time and under the same conditions.
(8) Embryo. The embryo is the small plant contained within a mature seed.
(9) FAME analysis. Fatty Acid Methyl Ester analysis is a method that allows for accurate quantification of the fatty acids that make up complex lipid classes.
(10) Imidazolinone resistance (Imi). Resistance and/or tolerance is conferred by one or more genes which alter acetolactate synthase (ALS), also known as acetohydroxy acid synthase (AHAS) allowing the enzyme to resist the action of imidazolinone.
(11) Mutagenesis. Mutagenesis refers to mutagenesis of a plant or plant part with a mutagen (e.g., a chemical or physical agent that increases the frequency of mutations in a target plant or plant part). By way of non-limiting example, the double chemical mutagenesis technique of Konzak, as described in U.S. Pat. No. 6,696,294 (the disclosure of which is incorporated by reference herein), can be used to induce mutant alleles in endogenous plant genes.
(12) Oil content. This is measured as percent of the whole dried seed and is characteristic of different varieties. It can be determined using various analytical techniques such as NMR, NIR, and Soxhlet extraction.
(13) Percentage of total fatty acids. This is determined by extracting a sample of oil from seed, producing the methyl esters of fatty acids present in that oil sample and analyzing the proportions of the various fatty acids in the sample using gas chromatography. The fatty acid composition can also be a distinguishing characteristic of a variety.
(14) Single Gene Converted (Conversion). Single gene converted (conversion) plant refers to plants which are developed by a plant breeding technique called backcrossing, or via genetic engineering, 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 or via genetic engineering.
(15) Stabilized. Reproducibly passed from one generation to the next generation of inbred plants of same variety.
(16) Total Saturated (TOTSAT). Total percent oil of the seed of the saturated fats in the oil including C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 and C24.0.
(17) According to a particular embodiment the invention, there is provided a novel sunflower plant producing seeds having low saturated fat content. This embodiment relates to the seeds of sunflower having low saturated fat content, to the plants, or plant parts, of sunflower plants producing seeds having low saturated fat content, and to methods for producing a sunflower plant produced by crossing the sunflower plant producing seeds having low saturated fat content with itself or another sunflower cultivar, and the creation of variants by mutagenesis or transformation of sunflower plants producing seeds having low saturated fat content.
(18) Other aspects of the invention provide novel sunflower plants producing seeds having low saturated fat content and high linoleic acid content. This embodiment relates to the seeds of sunflower having low saturated fat content and high linoleic acid content, to the plants, or plant parts, of sunflower plants producing seeds having low saturated fat content and high linoleic acid content, and to methods for producing a sunflower plant produced by crossing the sunflower plants producing seeds having low saturated fat content and high linoleic acid content with itself or another sunflower cultivar, and the creation of variants by mutagenesis or transformation of sunflower plants producing seeds having low saturated fat content and high linoleic acid content.
(19) Examples of seeds having low saturated fat content include, but are not limited to, seeds having about 2.8% or less, about 2.9% or less, about 3% or less, about 3.1% or less, about 3.2% or less, or about 3.3% or less total combined palmitic acid (16:0) and stearic acid (18:0) content.
(20) Examples of seeds of having low saturated fat content and high linoleic acid (18:2) content include, but are not limited to, seeds having about 6% or less, about 4.1% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less, about 10% or less, about 11% or less, or about 12% or less total combined palmitic acids (16:0) and stearic acid (18:0) content and having about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 74% or more linoleic acid (18:2).
(21) Thus, any such methods using the sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content, are part of this invention (e.g., selfing, backcrosses, hybrid production, crosses to populations, and the like). All plants produced using sunflower plants that produce seeds having as a parent low saturated fat and, optionally, high linoleic acid content, are within the scope of this invention. Advantageously, the sunflower plant could be used in crosses with other, different, sunflower plants to produce first generation (F.sub.1) sunflower hybrid seeds and plants with superior characteristics.
(22) In another aspect, the present invention provides for single or multiple gene converted sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content. 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, bacterial resistance, fungal resistance, viral disease resistance, male fertility, male sterility, enhanced nutritional quality, and industrial usage. The gene may be a naturally occurring sunflower gene or a transgene introduced through genetic engineering techniques.
(23) In another aspect, the present invention provides regenerable cells for use in tissue culture of sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content, and of regenerating plants having substantially the same genotype as the foregoing sunflower plant. The regenerable cells in such tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods or stems. Still further, an embodiment of the invention provides sunflower plants regenerated from the tissue cultures of the invention.
(24) In another aspect, the present invention provides a method of introducing a desired trait into sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content, wherein the method comprises: crossing a sunflower plant that produces seeds having low saturated fat and, optionally, high linoleic acid content with a plant of another sunflower cultivar that comprises a desired trait to produce F.sub.1 progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, and resistance to bacterial disease, fungal disease or viral disease; selecting one or more progeny plants that have the desired trait to produce selected progeny plants; crossing the selected progeny plants with the sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content to produce backcross progeny plants; selecting for backcross progeny plants that have the desired trait and physiological and morphological characteristics of sunflower plants that produce seeds having low saturated fat and, optionally, high linoleic acid content to produce elected backcross progeny plants; and repeating these steps to produce selected first or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of sunflower plants producing seeds having low saturated fat and, optionally, high linoleic acid content.
(25) Useful 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. Expression vectors can be introduced into plant tissues using the microprojectile media delivery with the biolistic device Agrobacterium-mediated transformation. Transformant plants obtained with the protoplasm of the invention are intended to be within the scope of this invention.
(26) With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode 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 genes, or additional or modified versions of native or endogenous genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as transgenes. Over the last fifteen to twenty years, several methods for producing transgenic plants have been developed and the present invention, in particular embodiments, also relates to transformed versions of the claimed variety or cultivar.
(27) Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA that includes 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 sunflower plants using transformation methods as described below to incorporate transgenes into the genetic material of the sunflower plant(s).
(28) Expression Vectors for Sunflower Transformation: Marker Genes
(29) 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.
(30) One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene under the control of plant regulatory signals, which confers resistance to kanamycin. See, e.g., Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin. See, e.g., Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).
(31) Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3-adenyl transferase and the bleomycin resistance determinant. See 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. See 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).
(32) Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. See 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).
(33) 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. See, R. A. Jefferson, Plant Mol. Biol. Rep. 5:387 (1987); Teeri et al., EMBO J. 8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987); DeBlock et al., EMBO J. 3:1681 (1984).
(34) Recently, in vivo methods for visualizing GUS activity that do not require destruction of plant tissue have been made available. Molecular Probes publication 2908, Imagene, T. M. 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.
(35) More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. See, Chalfie et al., Science 263:802 (1994). GFP and mutants of GFP may be used as screenable markers.
(36) Expression Vectors for Sunflower Transformation: Promoters
(37) 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 now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.
(38) As used herein, promoter includes reference to a region of DNA that is upstream from the start of transcription and that is 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 certain tissues 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 that 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.
(39) A. Inducible Promoters
(40) An inducible promoter is operably linked to a gene for expression in sunflower. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence that is operably linked to a gene for expression in sunflower. With an inducible promoter, the rate of transcription increases in response to an inducing agent.
(41) 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: those from the ACEI system that responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 gene from maize that responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen. Genetics 227:229-237 (1991); and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)); and Tet repressor from Tn10 (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. U.S.A. 88:0421 (1991).
(42) B. Constitutive Promoters
(43) A constitutive promoter is operably linked to a gene for expression in sunflower or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence that is operably linked to a gene for expression in sunflower.
(44) 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)); the promoters from rice actin genes (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 (1992)); 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, Xba1/NcoI fragment 5 to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to the Xba1/NcoI fragment), represents a particularly useful constitutive promoter. See PCT application WO 96/30530.
(45) C. Tissue-Specific or Tissue-Preferred Promoters
(46) A tissue-specific promoter is operably linked to a gene for expression in sunflower. 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 sunflower. Plants transformed with a gene of interest operably linked to a tissue-specific promoter can produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.
(47) 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 promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983), and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):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 Zm13 (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)).
(48) Transport of 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, can be 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.
(49) 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, e.g., Becker et al., Plant Mol. Biol. 20:49 (1992); P. S. Close, Master's Thesis, Iowa State University (1993); C. Knox et al., Structure and Organization of Two Divergent Alpha-Amylase Genes from Barley, Plant Mol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129 (1989); Fontes 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., A short amino acid sequence able to specify nuclear location, Cell 39:499-509 (1984); Steifel, et al., Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation, Plant Cell 2:785-793 (1990).
(50) Foreign Protein Genes and Agronomic Genes
(51) 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 that are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).
(52) In aspects of the invention, the transgenic plant provided for commercial production of foreign protein is a sunflower plant. In other aspects, 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. 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.
(53) Likewise, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes that may be used in this regard include, but are not limited to, those categorized below.
(54) 1. Genes that Confer Resistance to Pests or Disease and that Encode:
(55) 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 genes to engineer plants that are resistant to specific pathogen strains. See, e.g., Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); 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).
(56) B) A gene conferring resistance to a pest, such as soybean cyst nematode. See, e.g., PCT Application WO 96/30517; PCT Application WO 93/19181.
(57) C) A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, e.g., Geiser et al., Gene 48:109 (1986), which discloses the cloning and nucleotide sequence of a Bt ?-endotoxin gene. Moreover, DNA molecules encoding ?-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.
(58) D) A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.
(59) E) A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.
(60) F) An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, e.g., Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor); Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus .alpha.-amylase inhibitor); and U.S. Pat. No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).
(61) G) 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.
(62) H) An insect-specific peptide or neuropeptide, which 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), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., which discloses genes encoding insect-specific, paralytic neurotoxins.
(63) I) An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.
(64) J) An enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
(65) K) 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.
(66) L) 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.
(67) M) A hydrophobic moment peptide. See PCT application WO 95/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance).
(68) N) A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993), of heterologous expression of a cecropin-? lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.
(69) O) 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.
(70) P) 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 Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
(71) Q) 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.
(72) R) A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo ?-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-?-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436 (1992). The cloning and characterization of a gene that encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).
(73) S) 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.
(74) 2. Genes that Confer Resistance to an Herbicide:
(75) A) 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 AHAS 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.
(76) B) An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) or a benzonitrile (nitrilase gene). Przibila 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).
(77) 3. Genes that Confer or Contribute to a Value-Added Trait, Such as:
(78) A) Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).
(79) B) Decreased phytate content1) Introduction of a phytase-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) A gene could be introduced that reduced phytate content. In maize for example, this could be accomplished by cloning and then reintroducing DNA associated with the single allele that is responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).
(80) C) Modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteol. 170:810 (1988) (nucleotide sequence of Streptococcus mutants fructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacillus licheniformis ?-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 ?-amylase gene); and Fisher et al., Plant Physiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).
(81) Methods for Sunflower Transformation
(82) Numerous methods for plant transformation have been developed, including biological and physical plant transformation protocols. See, for example, Mild et al., Procedures for Introducing Foreign DNA into Plants in Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, 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, e.g., Gruber et al., Vectors for Plant Transformation in Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.
(83) A) Agrobacterium-mediated TransformationOne method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, e.g., Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, C. I. Kado, Crit. Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Mild et al., supra, and Moloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.
(84) B) Direct Gene TransferSeveral methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 ?m. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s, which is sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987); J. C. Sanford, Trends Biotech. 6:299 (1988); Klein et al., Bio/Technology 6:559-563 (1988); J. C. Sanford, Physiol. Plant 7:206 (1990); Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.
(85) Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J, 4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985), and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992), and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).
(86) Following transformation of sunflower target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods well known in the art.
(87) The foregoing methods for transformation would typically be used for producing a transgenic variety. The transgenic variety can then be crossed, with another (non-transformed or transformed) variety, in order to produce a new transgenic variety. Alternatively, a genetic trait, which has been engineered into a particular sunflower cultivar using the foregoing transformation techniques can be moved into another cultivar using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach can be used to move an engineered trait from a public, non-elite variety into an elite variety, or from a variety containing a foreign gene in its genome into a variety or varieties that do not contain that gene. As used herein, crossing can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
(88) Tissue Culture of Sunflowers
(89) Further production of a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content can occur by self-pollination or by tissue culture and regeneration. Tissue culture of various tissues of sunflower and regeneration of plants therefrom is known. For example, the propagation of a sunflower cultivar by tissue culture is described in U.S. Pat. No. 6,998,516.
(90) Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of soybeans and regeneration of plants therefrom is well known and widely published. For example, reference may be had to U.S. Pat. No. 6,998,516, which is incorporated herein in its entirety by reference. Thus, another aspect of this invention is to provide cells, which upon growth and differentiation, produce a sunflower plants having seeds containing low saturated fat and, optionally, high linoleic acid content.
(91) As used herein, the term tissue culture indicates a composition comprising isolated cells of the same or a different type, or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures include protoplasts, calli, plant clumps, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollen, flowers, seeds, pods, leaves, stems, roots, root tips, anthers, and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, 5,977,445, and 6,998,516 describe certain techniques, the disclosures of which are incorporated herein by reference.
(92) Single-Gene Converted (Conversion) Plants
(93) When the term sunflower plant is used in the context of the present invention, this also includes any single gene conversions of that variety. The term single gene converted plant as used herein refers to those sunflower plants that are developed by a plant breeding technique called backcrossing, or via genetic engineering, 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. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the variety. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to the recurrent parent (i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to the recurrent parent). The parental sunflower plant, which contributes the gene for the desired characteristic, is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental sunflower plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a sunflower plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent.
(94) The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single gene of the recurrent variety is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic and, therefore, the desired physiological and morphological constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross. One of the major purposes is to add some commercially desirable, agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.
(95) Many single gene traits have been identified that are not regularly selected for in the development of a new variety but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic, examples of these traits include but are not limited to, male sterility, waxy starch, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, male fertility, enhanced nutritional quality, industrial usage, yield stability and yield enhancement. These genes are generally inherited through the nucleus. Several of these single gene traits are described in U.S. Pat. Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which are hereby incorporated by reference.
(96) This invention also is directed to methods for producing a sunflower plant by crossing a first parent sunflower plant with a second parent sunflower plant, wherein the first or second parent sunflower plant is a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content. Further, both first and second parent sunflower plants can originate from a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content. Thus, any such methods using a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content are part of this invention (i.e., selfing, backcrosses, hybrid production, crosses to populations, and the like). All plants produced using a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content as a parent are within the scope of this invention, including those developed from varieties derived from a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content. Advantageously, the sunflower variety could be used in crosses with other, different, sunflower plants to produce first generation (F.sub.1) sunflower hybrid seeds and plants with superior characteristics. The variety of the invention can also be used for transformation where exogenous genes are introduced and expressed by the variety of the invention. Genetic variants created either through traditional breeding methods using a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content or through transformation of a sunflower plant producing seeds having low saturated fat and, optionally, high linoleic acid content by any of a number of protocols known to those of skill in the art are intended to be within the scope of this invention.
EXAMPLES
(97) The present invention is further described in the following examples, which are offered by way of illustration and are not intended to limit the invention in any manner.
Example 1: Sunflowers Producing Seeds Having Low Saturated Fat Content
(98) Sunflower germplasm with unusually low saturate levels has been developed through normal breeding techniques. Seed oil content of sunflower cultivars are provided in Table 1.
(99) TABLE-US-00001 TABLE 1 TOTAL C16:0 + Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS C18:0 H757B/LS10670B-B- 2.34 0.09 0.48 94.18 1.51 3.39 2.82 17-3-23.06 H757B/LS10670B-B- 2.47 0.11 0.51 93.62 2.11 3.42 2.98 17-3-33.11 H757B/LS10670B-B- 2.24 0.09 0.53 94.25 1.49 3.45 2.77 17-3-23.04 H757B/LS10670B-B- 2.70 0.13 0.50 93.26 2.24 3.67 3.2 17-3-02.08 H757B/LS10670B-B- 2.45 0.11 0.54 93.62 1.73 3.68 2.99 17-3-18.21 HE06EE010716.001 2.17 0.11 0.82 94.29 1.41 3.63 2.99 HE06EE010834.002 2.31 0.11 0.65 94.74 0.82 3.68 2.95 HE06EE010746.002 2.40 0.11 0.72 93.87 1.03 3.68 3.12 HE06EE010700.003 2.48 0.13 0.57 93.46 1.78 3.78 3.05 HE06EE016032.005 2.42 0.10 0.64 92.86 1.82 3.82 3.06 HE06EE016037.005 2.25 0.08 0.75 93.06 1.71 3.86 3.00 HE06EE016032.002 2.40 0.10 0.70 93.00 1.72 3.87 3.09 HE06EE010717.002 2.44 0.10 0.82 89.76 5.51 3.88 3.26 HE06EE010695.001 2.48 0.12 0.66 91.93 3.20 3.88 3.14 HE06EE010816.002 2.34 0.12 0.88 94.10 1.24 3.88 3.22 HE06EE010700.001 2.48 0.14 0.65 94.31 0.89 3.90 3.13 HE06EE010814.002 2.46 0.10 0.79 94.11 1.19 3.91 3.24 HE06EE010760.004 2.54 0.11 0.63 94.07 1.16 3.92 3.16 HE06EE010741.003 2.34 0.11 0.93 94.51 0.73 3.93 3.26 HE06EE010737.003 2.33 0.13 0.96 93.53 1.12 3.93 3.29 HE06EE016050.005 2.41 0.08 0.73 92.57 2.67 3.94 3.13 HE06EE016032.004 2.44 0.11 0.63 92.49 1.80 3.94 3.07 HE06EE010763.002 2.43 0.11 0.78 94.28 0.98 3.94 3.21 HE06EE010829.002 2.53 0.13 0.70 93.26 1.84 3.95 3.23 HE06EE010738.002 2.78 0.15 0.62 89.75 5.22 3.96 3.40 HE06EE010741.004 2.42 0.11 0.88 94.10 0.61 3.96 3.30 HE06EE010824.004 2.35 0.10 0.80 94.14 1.15 3.97 3.15 HE06EE010745.003 2.81 0.11 0.68 88.66 6.32 3.98 3.48 HE06EE010816.001 2.52 0.11 0.80 91.45 3.77 3.98 3.32
Example 2: Sunflowers Producing Seeds Having Low Saturated Fat Content and High Linoleic Acid Content
(100) Sunflower germplasm with unusually low saturate levels has been developed through normal breeding techniques. Seed oil content of sunflower cultivars are provided in Table 2.
(101) TABLE-US-00002 TABLE 2 TOTAL C16:0 + Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS C18:0 H757B/LS10670B-B- 4.25 0.09 1.13 37.87 55.45 5.90 5.38 17-3-14.01 H757B/LS10670B-B- 4.80 0.11 0.68 39.63 53.55 6.05 5.48 17-3-02.18 H757B/LS10670B-B- 4.01 0.08 1.37 38.48 54.68 6.07 5.38 17-3-27.12 H757B/LS10670B-B- 5.19 0.14 0.73 35.14 57.79 6.22 5.92 17-3-16.02 H757B/LS10670B-B- 4.99 0.09 1.25 17.97 74.37 6.81 6.24 17-3-36.22
Example 3: Sunflowers Producing Seeds Having Low Saturated Fat Content
(102) Sunflower germplasm with unusually low saturate levels has been developed through normal breeding techniques. Seed oil content of sunflower cultivars are provided in Table 3.
(103) TABLE-US-00003 TABLE 3 TOTAL Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS NuSun/No Saturate NS1982.16/OND163R-1-05 2.29 0.05 0.65 67.37 28.19 3.48 NS1982.8 2.09 0.08 0.55 79.40 15.99 3.10 No Saturate/High Oleic NS1982.8-03 1.60 0.03 0.37 95.13 1.48 2.33 NS1982.8 1.63 0.07 0.41 94.81 1.26 2.48 H117R[4]//H757B/LS10670B///NS 1.79 0.05 0.29 95.30 0.84 2.57 1982.6-2-023-1-12-076 Low Saturate/Linoleic CND117R/NS1982.8-3-06 5.29 0.07 0.73 18.19 74.43 6.41 OI1601B[2]//H757B/LS10670B[1]/// 3.76 0.07 0.80 34.97 58.62 5.29 NS1982.6 = B-3-04 CN2343B/4/CN2343B[2]//H757B/ 3.13 0.02 2.07 36.03 56.65 6.23 LS10670B///NS1982.11#1#1-3-11 Low Stearic NS1982.8/OND163R-2-12-009 2.75 0.66 0.25 92.95 1.99 3.43 H117R[4]//H757B/LS10670B/// 1.90 0.04 0.27 95.03 1.00 2.65 NS1982.6-2-023-1-12-038 OID263R/NS1982.8-4-12-002 3.08 0.12 0.27 93.54 1.48 3.87 Low Palmitic H251B[2]/IAST-4 = 1 = 100// 1.47 0.24 2.59 92.59 0.65 5.42 NS1982.16-11-39-041 NS1982.14-08 1.51 0.02 2.24 92.84 1.35 4.90 NS1982.16 1.52 0.06 1.05 94.37 0.85 3.39 Very High Oleic H117R[4]//H757B/LS10670B// 1.79 0.05 0.29 95.30 0.84 2.57 NS1982.6-2-023-1-12-076 NS1982.8/OND163R-2-12-059 1.87 0.10 0.44 95.22 0.97 2.76 ON3351B/NS1982.8-1-04 2.04 0.03 0.50 95.20 0.70 3.08
(104) As can be seen in Table 3, the data demonstrates seed oil having total saturates as low as 2.33% in a high oleic (>80%) background, no Saturate (<3.5%) profile in a NuSun (55-50% oleic) background, oleic levels up to 95.30%; stearic levels as low as 0.25%, palmitic levels as low as 1.47%, and low Saturate (<7.0%) profile in a linoleic (<55% oleic) background.
Example 4: Sunflowers Producing Seeds Having Low Saturated Fat, Stearic Acid, and Palmitic Acid Content
(105) Sunflower germplasm with unusually low saturate levels has been developed through normal breeding techniques. Seed oil content of sunflower cultivars are provided in Table 4.
(106) TABLE-US-00004 TABLE 4 Name C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 NS1982.8/OND163R-12-90 1.37 0.01 1.70 91.93 2.83 0.08 0.21 0.60 H117R[4]//H757B/ 1.39 0.02 0.53 94.89 1.55 0.08 0.09 0.66 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ 1.44 0.03 0.36 94.83 1.84 0.09 0.08 0.74 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ 1.58 0.02 0.24 94.54 2.05 0.10 0.06 0.79 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ 1.89 0.03 0.24 94.17 2.31 0.13 0.04 0.70 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ 1.94 0.03 0.23 94.58 1.80 0.12 0.07 0.69 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 Name C20:2 C22:0 C22:1 C24:0 C24:1 TSats NS1982.8/OND163R-12-90 nd 0.70 0.01 0.33 nd 4.32 H117R[4]//H757B/ nd 0.40 0.03 0.19 nd 2.60 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ nd 0.31 0.02 0.14 nd 2.33 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ nd 0.24 0.04 0.15 nd 2.28 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ nd 0.21 0.03 0.11 nd 2.50 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/ nd 0.22 0.03 0.13 nd 2.60 LS10670B-B-17-3-23 = B1 = 2 = 16///NS1982.6-2-23.1-1
(107) As can be seen in Table 4, this set of data includes the low values for stearic (0.23%), palmitic (1.37%), and total saturated oils (2.28%).
Example 5: Marker Development for Low Stearic and Low Palmitic
(108) A strategy for marker development was developed as described herein. First, markers from the target QTL regions, developed at Dow AgroSciences as well as from the public resources, were identified and screened for polymorphisms between the parental lines of corresponding mapping populations. Polymorphic markers were then screened in the mapping populations. For monomorphic (non-informative) markers, primers were designed to amplify their corresponding genomic loci and the amplicons were sequenced to identify single nucleotide polymorphisms (SNPs), if any, between the parental lines. TaqMan MGB Allelic Discrimination assays were developed for the identified SNPs and were mapped on the respective population. Second, based on sequences of candidate genes for fatty acids, primers flanking introns were designed to isolate fatty acid gene sequences from the parental lines. Nucleotide polymorphisms at the sequence level were developed into markers based on their polymorphic nature and were then screened in the mapping populations. JoinMap 3.0 (Van Ooijen, 2004a) was employed to map the newly developed markers, and MapQTL 5 (Van Ooijen, 2004b) was used to fine map QTLs.
(109) A) Marker Development for Low Stearic Acid
(110) SSR marker development: Eight SSR markers were screened for polymorphisms between parental lines ONN687R and H757B/LS10760B-B-17-3-23-5 of the ONN687R?H757B/LS10760B-B-17-3-23-5 mapping population that was previously used to map the target low stearic acid QTL. (See Table 5.) Four SSR markers, HA0442, CRT22, ORS565 and ORS732, were polymorphic. HA0442 and CRT22 amplicons from ONN687R and H757B/LS10760B-B-17-3-23-5 were resolved on ABI 3730 sequencer, and were 163 bp and 165 bp, respectively, for marker HA0442, and 290 bp and 261 bp, respectively, for CRT22. ORS565 and ORS732 amplicons from ONN687R and H757B/LS10760B-B-17-3-23-5 were resolved on 3% Metaphor gels. The corresponding mapping population ONN687R?H757B/LS10760B-B-17-3-23-5 was genotyped with HA0442, CRT22, ORS565 and ORS732 using the following PCR primers and reaction conditions.
(111) TABLE-US-00005 HA0442ForwardPrimer: (SEQIDNO:1) 5-HEX-TGGAACTGTAAATGGACCCAAG-3 HA0442ReversePrimer: (SEQIDNO:2) 5-GCACTGCACCATTTATGAGAAG-3 CRT22ForwardPrimer: (SEQIDNO:3) 5-HEX-TCGAGATGAAACCGAATGAAGAAA-3 CRT22ReversePrimer: (SEQIDNO:4) 5-GTTTCTTGGGACTGATATTGCCAAGTGGG-3 ORS565ForwardPrimer: (SEQIDNO:5) 5-TGGTCAACGGATTTAGAGTCAA-3 ORS565ReversePrimer: (SEQIDNO:6) 5-TCCAGTTTGGTCTTGATTTGG-3 ORS732ForwardPrimer: (SEQIDNO:7) 5-GCACGGAACTCCTCAAATGT-3 ORS732ReversePrimer: (SEQIDNO:8) 5-GCACGGGAAACAAAGAGTCA-3
(112) PCR Components: 4 ng gDNA 1?PCR buffer (Qiagen, Valencia, Calif.) 0.25 ?M Forward primer 0.25 ?M Reverse primer 1 mM MgCl.sub.2 0.1 mM of each dNTP 0.4% PVP 0.04 Units HotStar Taq DNA polymerase (Qiagen, Valencia, Calif.) Total Volume: 4.8 ?l
(113) Thermocycler Setup: Step 1: 94? C. for 12 minutes Step 2: 94? C. for 30 seconds Step 3: 55? C. for 30 seconds Step 4: 72? C. for 30 seconds Step 5: repeat steps 2, 3 and 4 for 35 cycles Step 6: 72? C. for 30 minutes
(114) SNP marker development: Eight pairs of primers were used to amplify eight genomic loci from both ONN687R and H757B/LS10760B-B-17-3-23-5 to develop SNP markers (Table 6). Three primer pairs (ZVG76snpF/R, ZVG77snpF/R, and ZVG78snpF/R) were designed based on sequences from restriction fragment length polymorphism (RFLP) probes ZVG76, ZVG77 and ZVG78 (Kolkman et al., 2007). Primer sequences for HT57F/R, HT64F/R, HT131F/R, HT134F/R, and HT210F/R were from Lai et al., (2005). SNPs were found in the amplicons from HT64F/R, HT210F/R, and ZVG78snpF/R. TaqMan MGB Allelic Discrimination assays were developed for one SNP locus in the HT64F/R amplicon and one SNP locus in the ZVG78snpF/R amplicon (See below), and the ONN687R?H757B/LS10760B-B-17-3-23-5 mapping population was genotyped with those two SNP markers using the developed TaqMan assays.
(115) There were four SNP loci (marked in bold) in the HT64F/R amplicons from ONN687R and H757B/LS10760B-B-17-3-23-5. The TaqMan Assay was developed for the R-locus. The sequences for Forward Primer, Reverse Primer, Probe 1 and Probe 2 are 5-CCGGCTGCTTCTAGACCTTATAAG-3 (SEQ ID NO:9), 5-TCGTCGGTGGGACACACA-3 (SEQ ID NO:10), 5-6FAM-ACTGTTGGATCGGTTC-3 (SEQ ID NO:11), and 5-VIC-CACTGTTGGATCGATT-3 (SEQ ID NO:12), respectively.
(116) TABLE-US-00006 (SEQIDNO:13) TTATTCTCGGCTTCCGGTGTGATTTTACTCTCATGGTTAAGTTTTCAAGA GATTGTCGCY(T/C)GCTGAAAACTTTTTATATTGTTTCGGTATGATCTT GGAGTTTATAGCCTTTGTAAGGTTAAGAATGAAACACCCGGCTGCTTCTA GACCTTATAAGATACCCGTGGGCACTGTTGGATCGR(A/G)TTCTTCTGT GTGTCCCACCGACGATTTTGATCTGTGTCGTGTTGGCTCTTTCTTCACTC AAGGTCATGATCGTTAGY(T/C)GTY(C/T)ATTGCCATATTTTTCGGGT TCGCATTGCAACCGTTTTTAAAGTTTGCCGAGAAGAAAAGATGGCTTAAA TTTTCAACTAAAGCCGATCTTCCCG
(117) There were also four SNP loci (marked in bold) in the ZVG78snpF/R amplicons from ONN687R and H757B/LS10760B-B-17-3-23-5. The TaqMan Assay was developed for the R-locus at the 5 end. The sequences for Forward Primer, Reverse Primer, Probe 1 and Probe 2 are 5-GTCCATCTTTCCTCAACGACTTG-3 (SEQ ID NO:14), 5-CCTAAACGCCTCGAAAAAGCT-3 (SEQ ID NO:15), 5-6FAM-TTACCATGTCTATAATGC-3 (SEQ ID NO:16), and 5-VIC-ATTACCATGTCTGTAATGC-3 (SEQ ID NO:17), respectively.
(118) TABLE-US-00007 (SEQIDNO:18) AACTGAGTTCTGTACGCCAGAGATTTGCCCGACCATGACCGCAGGTCCAA AGTAAGTCTTGCTATTGCACATTTGCACGATTAACGGTTTCTTATATAGA AGATACATGATTCTTGAATTTATGTAAATAAAACTTGACAGATATGAATA CCGATGGGCTGATGGTGTGCAAATCAAGAAGCCTATTGAAGTTTCGGCTC CAAAGTACGTAGAGTTCTTGATGGATTGGATTGAGTCACAATTGGATGAC GAGTCCATCTTTCCTCAACGACTTGGTAATTAGTTAATTACCATGTCTR (G/A)TAATGCATCATTTAATAAAGCTTTTTCGAGGCGTTTAGGAAACTG AAATAGTAATTTTCGATTGY(T/C)CGTGCAGGAGCGCCATTTCCCGCCA ATTTTAGGGACGTTGTGAAAACGATATTTAAACGCTTGTTTCGTGTATAY (T/C)GCGCATATCTACCACACR(G/A)CATTTTCAGAAGATTGTGAGTC TTAAAGAAGAAGCCCATCTAAACACTTGTTTCAAGCATTTCATATTGTTT ACATGTGTAA
(119) The following PCR setup was used for both SNP markers.
(120) Real-Time PCR Components: 25 ng gDNA 1? Taqman Universal PCR Master Mix 22.5 ?M Forward Primer 22.5 ?M Reverse Primer 5 ?M Probe 1 5 ?M Probe 2 Total Volume: 25 ?l
(121) Bio-Rad iCycler Setup: Step 1: 95? C. for 15 minutes Step 2: 94? C. for 30 seconds Step 3: 60? C. for 1 minute Step 4: repeat steps 2 and 3 for 65 cycles Step 5: 4? C. forever
(122) Indel marker development: Primers were designed to amplify and sequence 32 fatty acid related genes from the two parental lines ONN687R and H757B/LS10760B-B-17-3-23-5. Seven genes had polymorphisms, four genes had weak amplifications, and all others were monomorphic (Table 6). The mapping population ONN687R?H757B/LS10760B-B-17-3-23-5 was screened with all identified polymorphisms.
(123) Mapping new markers and fine mapping low stearic acid QTL: JoinMap 3.0 (Van Ooijen, 2004a) was used to map all newly identified polymorphic markers. Marker CRT22 gave a significant segregation distortion and was not mapped. Six markers developed from the candidate gene approach mapped to chromosomes other than the target chromosome 17 (Table 6). Seven markers HA0442, ORS565, HT64, ZVG78, KASI-2, KASI-4, and ORS732 were mapped to chromosome 17. Fatty acid genes KASI-2 and KASI-4 were mapped to chromosome 17 but not close to the target low stearic acid QTL (
(124) B) Developing and Mapping an Indel Marker for Palmitic Acid QTL
(125) SNPs and indels were observed in the amplicon sequences of parental lines H280R[1]/687R-1-8-1 and OND163R with the primer pair for fatty acid gene KASIII-2 (Table 6,
(126) TABLE-US-00008 TABLE5 ListofmarkersinvestigatedtosaturatethelowstearicacidQTLregion. FName Sequence RName Sequence Note 1)SSR HA0953F-HEX CAAACCAACAACCACCATCA HA0953R AAACGACACCGATGAGAACC Monomorphic (SEQIDNO:34) (SEQIDNO:35) HA1909F-FAM CTGAGTTTCGTGTACCATTTCTATTG HA1909R ACACCAATCAGTGGGTTTCATC Poormarker (SEQIDNO:36) (SEQIDNO:37) HA0442F-HEX TGGAACTGTAAATGGACCCAAG HA0442 GCACTGCACCATTTATGAGAAG Polymorphic (SEQIDNO:1) (SEQIDNO:2) CRT22F-HEX TCGAGATGAAACCGAATGAAGAAA CRT22R GTTTCTTGGGACTGATATTGCCAAGTGGG Polymorphic (SEQIDIDNO:3) (SEQIDNO:4) ORS297F-FAM TGCAAAGCTCACACTAACCTG ORS297R GTGTCTGCACGAACTGTGGT Monomorphic ZVG76ssrF-FAM GCACCCTAGAGCTTCATTCG ZVG76ssrR AGCCCAAGGATGTTGTTTTG Monomorphic ORS565F TGGTCAACGGATTTAGAGTCAA ORS565R TCCAGTTTGGTCTTGATTTGG Polymorphic (SEQIDNO:5) (SEQIDNO:6) ORS732F GCACGGAACTCCTCAAATGT ORS732R GCACGGGAAACAAAGAGTCA Polymorphic (SEQIDNO:7) (SEQIDNO:8) 2)SNP HT57F GCGATTATTGTTATGGACGC HT57R AGCGGAAACTGTTCTTGTTG Monomorphic (SEQIDNO:19) (SEQIDNO:20) HT64F TTATTCTCGGCTTCCGGT HT64R CGGGAAGATCGGCTTTAG SNPs (SEQIDNO:21) (SEQIDNO:22) HT131F CGTAACATGCAAGTTGTGGA HT131R TGTACTCTAAACGGGCAACC Monomorphic (SEQIDNO:23) (SEQIDNO:24) HT134F AGTCATGCTTGAAGGAGCTG HT134R CTCTGTCAGCTTGCAATGAA Monomorphic (SEQIDNO:25) (SEQIDNO:26) HT210F CTAAAACTGTCGCAAGGGAA HT210R CCTCCATCAATGGTAAGCAC SNPs (SEQIDNO:27) (SEQIDNO:28) ZVG76snpF TCCAACTCATGAACGGACTCT ZVG76snpR SameasZVG76ssrR Monomorphic (SEQIDNO:29) ZVG77snpF TTGGTGACTCTTGCAGCATC ZVG77snpR AAGTTTAAAACCGCGTCGTG Monomorphic (SEQIDNO:30) (SEQIDNO:31) ZVG78snpF TATGAGCCTCTTCGGTCTCG ZVG78snpR CACCTTATTCAGCCCCGAT SNPS (SEQIDNO:32) (SEQIDNO:33)
(127) TABLE-US-00009 TABLE 6 Fatty acids genes investigated. Marker Enzyme Results on stearic population Map position KASIII-1 Ketoacyl-ACP Syntetase III Co-dominant polymorphism LG5 KASIII-2 Ketoacyl-ACP Syntetase III Non-polymorphic KASIII-3 Ketoacyl-ACP Syntetase III Weak amplification KASI-1 Ketoacyl-ACP Syntetase I Non-polymorphic KASI-2 Ketoacyl-ACP Syntetase I Co-dominant polymorphism LG17 KASI-4 Ketoacyl-ACP Syntetase I Co-dominant polymorphism LG17 KASI-3 Ketoacyl-ACP Syntetase I Weak amplification KASII-1 Ketoacyl-ACP Syntetase II Non-polymorphic KASII-2 Ketoacyl-ACP Syntetase II Weak amplification KASII-3 Ketoacyl-ACP Syntetase II Co-dominant polymorphism LG9 KAR Ketoacyl reductase Non-polymorphic HAD Hyroxyacyl-ACP dehydratase Non-polymorphic Ear1 Enoyl-ACP reductase Non-polymorphic FATA-1 FATA thioesterase Non-polymorphic FATA-2 FATA thioesterase Non-polymorphic FATA-3 FATA thioesterase Dominant polymorphism LG7 FATB-1 FATB thioesterase Weak amplification FATB-2 FATB thioesterase Non-polymorphic CT-alpha1 ACC?>carboxyltransferase-alpha (accA) Dominant polymorphism LG10 BCCP ACC?>biotin carboxyl carrier protein (aacB) Non-polymorphic KCS1 Ketoacyl-CoA synthase-I Non-polymorphic KCS2 Ketoacyl-CoA synthase-II Non-polymorphic KCS3 Ketoacyl-CoA synthase-III Non-polymorphic SAD 17 Stearoyl-ACP desaturase Co-dominant polymorphism LG1 erLPAT Lysophosphatidic acid acyl transferase Non-polymorphic erPAP Phophatidic acid acyl transferase Non-polymorphic PDPS Phosphatidylglycerophosphatase synthase Non-polymorphic erLDS ER linoleate desaturase Non-polymorphic FAD6-1 Plastid oleate desaturase Non-polymorphic FAD6-2 Plastid oleate desaturase Non-polymorphic FAD2-1F5-R2 Oleate desaturase Non-polymorphic FAD2-1F5-R3 Oleate desaturase Non-polymorphic
(128) While this invention has been described in certain embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.