# Patent

## Title
Acetyl-CoA carboxylase herbicide resistant sorghum

## Abstract

The present invention provides for compositions and methods for producing crop plants that are resistant to herbicides. In particular, the present invention provides for sorghum plants, plant tissues and plant seeds that contain altered acetyl-CoA carboxylase (ACC) genes and proteins that are resistant to inhibition by herbicides that normally inhibit the activity of the ACC protein.

## Background
The present application claims priority to U.S. Provisional Application No. 60/880,125 filed Jan. 12, 2007, incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention provides for compositions and methods for producing crop plants that are resistant to herbicides. In particular, the present invention provides for sorghum plants, plant tissues and plant seeds that contain altered acetyl-CoA carboxylase (ACC) genes and proteins that are resistant to inhibition by herbicides that normally inhibit the activity of the ACC protein.
BACKGROUND OF THE INVENTION
Sorghum is the second most important cereal-feed grain grown in the United States. Production is economically critical to farms operating in marginal rainfall areas because of sorghum's ability to tolerate drought and heat. Both the livestock and bio-energy industries utilize sorghum as an energy substrate thereby making it a versatile crop.
Worldwide, sorghum is the fifth leading cereal grain. As it is tolerant to both drought and heat, it is easily the most widely grown food grain in the semiarid regions of sub-Sahelian Africa and in the dry central peninsular region of India. As such, sorghum is used in human consumption in most of the driest regions of the world thereby making it a critically important food crop in these locations.
The development of herbicide resistance in plants offers significant production and economic advantages; as such the use of herbicides for controlling weeds or plants in crops has become almost a universal practice. However, application of such herbicides can also result in death or reduced growth of the desired crop plant, making the time and method of herbicide application critical or in some cases unfeasible.
Of particular interest to farmers is the use of herbicides with greater potency, broad weed spectrum effectiveness and rapid soil degradation. Plants, plant tissues and seeds with resistance to these compounds would provide an attractive solution by allowing the herbicides to be used to control weed growth, without risk of damage to the crop. One such class of broad spectrum herbicides are those compounds that inhibit the activity of the acetyl-CoA carboxylase (ACC) enzyme in a plant. Such herbicides are included in the aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemical families. For example, sorghum is susceptible to many ACC inhibiting herbicides that target monocot species, making the use of these herbicides to control grassy weeds almost impossible.
Certain weed grass species have been found that display altered sensitivity to FOP and DIM herbicides. One grass species, black grass (A. myosuroides [Huds.]), is a major grass weed in Europe. Several mutations have been found in the genome of some black grass plants that confer resistance to some, but not all, FOP and DIM herbicides (Délve, et al., 2005, Plant Phys. 137:794-806; Délye, et al., 2002, Theor. Appl. Genet. 104:1114-1120). Similar findings were found in mutant grass weeds such as annual ryegrass (L. rigidum [Gaud.]; Délye, et al., 2002, Pest Manag. Sci. 58:474-478), green foxtail (S. viridis [L. Beauv.]; Zhang and Devine, 2000, Weed Sci. Soc. Am. 40:33; Délye, et al., 2002, Planta 214:421-427) and wild oat (A. fatua [L.]; Christoffers et al., 2002, Genome 45:1049-1056). One herbicide resistant maize hybrid (DK592 from Dekalb) has a similar mutation in the ACC enzyme as that found in grass weeds (Zagnitko et al., 2001, Proc. Natl. Acad. Sci. 98:6617-22).
Due to the importance of sorghum as a crop plant on the world stage, what are needed are sorghum hybrids that are resistant to the inhibitory effects of ACC herbicides, thereby allowing for greater crop yield when these herbicides are used to control grassy weeds.
SUMMARY OF THE INVENTION
The present invention provides for compositions and methods for producing sorghum cultivars that are resistant to herbicides. In particular, the present invention provides for sorghum plants, plant tissues and plant seeds that contain altered acetyl-CoA carboxylase (ACC) genes and proteins that are resistant to inhibition by herbicides that normally inhibit the activity of the ACC protein.
Cultivated sorghum [Sorghum bicolor (L.) Moench] is susceptible to many ACC inhibiting herbicides that target monocot or grassy weed species. However, as described herein a sorghum genotype was found that exhibits tolerance to ACC inhibiting herbicides. Genetic analysis has identified genetic differences within a wild sorghum germplasm that results in an ACC herbicide resistance phenotype.
In one embodiment, the present invention provides for one or more sorghum plants whose germplasm comprises a mutation that renders the plant tolerant to ACC herbicides. Moreover, in further embodiments the invention relates to the offspring (e.g., F1, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for sorghum hybrids whose germplasm contains a mutation, such that the phenotype of the plants is ACC herbicide resistance. In some embodiments, said offspring (e.g., F1, F2, F3, etc.) are the result of a cross between elite sorghum lines, at least one of which contains a germplasm comprising a mutation that renders the plant tolerant to ACC herbicides.
In one embodiment, the present invention provides a sorghum hybrid wherein said sorghum hybrid germplasm confers resistance to inhibition by one or more acetyl-CoA carboxylase herbicides at levels of said one or more herbicides that would normally inhibit the growth of a sorghum hybrid. In some embodiments, said one or more acetyl-CoA carboxylase herbicides are from aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemical families. In some embodiments, said sorghum hybrid germplasm that confers resistance to inhibition by one or more acetyl-CoA carboxylase herbicides comprises one or more mutations in the acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or as found in ATCC No. PYA-8034.
In one embodiment, the present invention provides a method of controlling weeds in the vicinity of a sorghum hybrid, comprising providing one or more acetyl-CoA carboxylase herbicides, applying said one or more acetyl-CoA carboxylase herbicides to a field comprising a sorghum hybrid, and controlling weeds in the vicinity of said sorghum hybrid such that weed growth is adversely affected by the application of said one or more herbicides and growth of said sorghum hybrid is not adversely affected. In some embodiments, said one or more acetyl-CoA carboxylase herbicides are from aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemical families. In some embodiments, said sorghum hybrid comprises one or more mutations in the acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034.
In one embodiment, the present invention provides a sorghum hybrid, wherein said sorghum hybrid comprises a germplasm comprising one or more mutations in the acetyl-CoA carboxylase gene such that resistance to one or more acetyl-CoA carboxylase herbicides is conferred to said hybrid. In some embodiments, said sorghum hybrid is created by introgression of a sorghum germplasm that comprises said one or more mutations for conferring resistance to one or more acetyl-CoA carboxylase herbicides. In some embodiments, said sorghum hybrid is created by incorporation of a heterologous gene comprising one or more mutations for conferring resistance to one or more acetyl-CoA carboxylase herbicides.
In one embodiment, the present invention provides a method for producing a sorghum hybrid plant line resistant to one or more acetyl-CoA carboxylase herbicides comprising identifying a germplasm conferring said herbicide resistance, wherein said herbicide resistant germplasm derives from an herbicide resistant sorghum plant, and introducing said germplasm into an elite sorghum plant line. In some embodiments, said introducing of said germplasm into said elite sorghum plant line is by introgression. In some embodiments, said introducing of said germplasm into said elite sorghum plant line is by introduction of a heterologous gene.
In one embodiment, the present invention provides a sorghum hybrid wherein the germplasm of said hybrid comprises conferred resistance to one or more acetyl-CoA carboxylase herbicides and resistance to one or more compounds from one or more herbicide groups that are not acetyl-CoA carboxylase inhibitors.
In one embodiment, the present invention provides a method for identifying sorghum plant lines resistant to acetyl-CoA carboxylase herbicides comprising supplying a nucleic acid sample from a sorghum plant, providing amplification primers for amplifying a region of a sorghum plant corresponding to an acetyl-CoA carboxylase gene present in said nucleic acid sample, applying said amplification primers to said nucleic acid sample such that amplification of said region of said acetyl-CoA carboxylase gene occurs, and identifying sorghum plants resistant to acetyl-CoA carboxylase herbicides based on the presence of one or more mutations that confer acetyl-CoA carboxylase herbicide resistance present in said amplified nucleic acid sample.
In one embodiment, the present invention provides for sorghum seeds wherein said germplasm of said seeds comprises a mutant acetyl-CoA carboxylase gene such that said mutation confers resistance to inhibition by acetyl-CoA carboxylase herbicides. In some embodiments, the germplasm of said sorghum seeds comprise a mutant acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034. In some embodiments, the present invention provides for sorghum plants that grow from said seeds and further plant parts that comprise said sorghum plants grown from said seeds. In some embodiments, the mutant acetyl-CoA carboxylase gene is a functional fragment of the gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034, such that the gene fragment encodes a protein fragment that is sufficient to confer resistance to inhibition by acetyl-CoA carboxylase herbicides to a sorghum plant. In some embodiments, the present invention provides for sorghum plants that grow from said seeds and further plant parts that comprise said sorghum plants grown from said seeds.
In some embodiments, the present invention provides for a sorghum hybrid that comprises a gene that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034. In some embodiments, the acetyl-CoA carboxylase herbicide resistance gene that is as least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to SEQ ID NO: 12 as found in ATCC No. PTA-8033 and/or ATCC No. PYA-8034 comprises one or more amino acid substitutions, for example, a tryptophan to cysteine amino acid substitution at an amino acid position aligning with Trp7027 of the A. myosuroides Huds. ACC protein as found in SEQ ID NO: 12.
In one embodiment, the present invention further provides for sorghum hybrid plants that have all the physiological and morphological characteristics of said sorghum plant grown from said sorghum seed. In further embodiments, the present invention provides for tissue cultures and regenerated tissue cultures that arise from said sorghum seed or said sorghum plant part that comprises a mutation in said acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034.
In one embodiment, the present invention provides a method of producing sorghum seed comprising crossing a plant comprising a mutant acetyl-CoA carboxylase gene as found in ATCC No. PTA-8033 or ATCC No. PYA-8034 with itself or a second sorghum plant and collecting said seed from said cross. In some embodiments, the methods for producing said sorghum seed comprises planting a parent seed sorghum line wherein said parent seed line comprises a germplasm that confers resistance to acetyl-CoA carboxylase herbicides with a parent pollinator sorghum line wherein said pollinator and/or seed line germplasm comprises a germplasm that confers resistance to acetyl-CoA carboxylase herbicides, growing said parent seed and pollinator sorghum plants together, allowing for the said parent seed plants to be pollinated by said parent pollinator plants, and harvesting the seed that results from said pollination.



DESCRIPTION OF THE FIGURES
FIG. 1 shows the mutation of tryptophan (TGG) to cysteine (TGC) in the sorghum ACC gene found to be associated with ACC herbicide resistance. Sorghum line Bol-71 ACC gene (bases 977-1017)—SEQ ID NOs: 2 and 9. Sorghum line R91 ACC gene (bases 977-1017)—SEQ ID NOs: 3, 8, and 10. Sorghum line Bol-36 ACC gene (bases 977-1017)—SEQ ID NOs: 4 and 7. Sorghum line ATx623 ACC gene (bases 977-1017)—SEQ ID NOs: 5 and 11. Sorghum line Tx430 ACC gene (bases 977-1017)—SEQ ID NO:6.



DEFINITIONS
As used herein, the term “variety” and “cultivar” refers to plants that are defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, distinguished from any other plant grouping by the expression of at least one of the characteristics and considered as a unit with regard to its suitability for being propagation unchanged.
As used herein, the term “hybrid” refers to the offspring or progeny of genetically dissimilar plant parents or stock produced as the result of controlled cross-pollination as opposed to a non-hybrid seed produced as the result of natural pollination.
As used herein, the term “progeny” refers to generations of a plant, wherein the ancestry of the generation can be traced back to said plant.
As used herein, the term “derivative” of an herbicide resistant plant includes both the progeny of that herbicide resistant plant, as well as any mutant, recombinant, or genetically engineered derivative of that plant, whether of the same species or a different species, where the herbicide resistant characteristic(s) of the original herbicide resistant plant has been transferred to the derivative plant.
As used herein, the term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, cellus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
As used herein, the term “plant part” as used herein refers to a plant structure or a plant tissue, for example, pollen, an ovule, a tissue, a pod, a seed, and a cell. In some embodiments of the present invention transgenic plants are crop plants.
As used herein, the terms “crop” and “crop plant” are used in their broadest sense. The term includes, but is not limited to, any species of plant edible by humans or used as a feed for animal or fish or marine animal, or consumed by humans, or used by humans, or viewed by humans, or any plant used in industry or commerce or education.
As used herein, the terms “F-generation” and “filial generation” refers to any of the consecutive generations of plants, cells, tissues or organisms after a biparental cross. The generation resulting from a mating of the a biparental cross (i.e. two parents) is the first filial generation (designated as “F1” and “F1”) in reference to a seed and it's plant, while that resulting from crossing of F1 individual is the second filial generation (designated as “F2” or “F2”) in reference to a seed and it's plant. For example, an F2 seed and a resulting plant are produced by self-pollination or cross-pollination of F1, while later F generations are produced from self-pollination or cross-pollination of the immediate prior generation.
As used herein, the term “germplasm” refers to any genetic material of plants that contain functional units of heredity.
As used herein, the term “elite germplasm” in reference to a plant refers to hereditary material of proven genetic superiority.
As used herein, the term “elite plant,” refers to any plant that has resulted from breeding and selection for superior agronomic performance.
As used herein, the term “trait” refers to an observable and/measurable characteristic of an organism. For example, the present invention describes plants that are resistant to FOP and DIM herbicides.
As used herein, the terms “marker” and “DNA marker” and “molecular marker” in reference to a “selectable marker” refers to a physiological or morphological trait that may be determined as a marker for its own selection or for selection of other traits closely linked to that marker. For example, such a marker could be a gene or trait that associates with herbicide tolerance including, but not limited to, simple sequence repeat (SSR), single nucleotide polymorphism (SNP), genetic insertions and/or deletions and the like.
As used herein, the term “introgress” and “introgressing” and “introgression” refers to conventional (i.e. classic) pollination breeding techniques to incorporate foreign genetic material into a line of breeding stock. For example, the present invention provides for sorghum crop plants introgressed with a mutant ACC gene for herbicide tolerance by crossing two plant generations.
As used herein, the term “wild-type” when made in reference to a gene refers to a functional gene common throughout a plant population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
As used herein, the terms “modified” or “mutant” or “functional mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Thus, the terms “modified” and “mutant” when used in reference to a nucleotide sequence refer to an nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide acid sequence and the term “functional mutant” when used in reference to a polypeptide encodes by said “modified” or “mutant” nucleic acid refers to the protein or polypeptide that retains activity. In the present application, the ACC mutant protein, “or functional mutant” thereof is an ACC gene that retains its native activity to create essential amino acids. Additionally, a “modified” nucleotide sequence is interpreted as that found in the degenerate genetic code as known by those skilled in the art. For example, the genetic code is degenerate as there are instances in which different codons specify the same amino acid; a genetic code in which some amino acids may each be encoded by more than one codon. It is contemplated that the present invention may comprise such degeneracy (e.g., wherein a sorghum hybrid comprises an ACC gene that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to SEQ ID NO: 1) as found in, for example, the sorghum germplasm.
As used herein, the term “heterologous” when used in reference to a gene or nucleic acid refers to a gene that has been manipulated in some way.
As used herein, the term “portion” or “functional fragment” when used in reference to a protein (as in “a fragment of a given protein”, “a protein fragment”, a “portion of a protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino sequence minus one amino acid. In the present invention, the protein fragment is preferentially functional such that the protein fragment confers resistance to inhibition to ACC herbicides to a given plant.
DETAILED DESCRIPTION OF THE INVENTION
Acetyl-CoA carboxylase (ACC) is a biotinylated enzyme that catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA. This carboxylation is a two-step, reversible reaction consisting of the ATP-dependent carboxylation of the biotin group on the carboxyl carrier domain by biotin-carboxylase activity followed by the transfer of the carboxyl group from biotin to acetyl-CoA by carboxyl-transferase activity (Nikolau et al., 2003, Arch. Biochem. Biophys. 414:211-22). Acetyl-CoA carboxylase is not only a key enzyme in plants for biosynthesis of fatty acids, a process that occurs in chloroplasts and mitochondria, but ACC also plays a role in the formation of long-chain fatty acids and flavonoids, and in malonylation that occurs in the cytoplasm. There are two isoforms of ACC with the chloroplastic ACC accounting for more than 80% of the total ACC activity (Herbert et al., 1996, Biochem. J. 318:997-1006). Aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) are two classes of chemicals that are known to selectively inhibit chloroplastic ACC in grasses (Rendina et al., 1990, J. Agric. Food Chem. 38:1282-1287).
Seeds from 83 wild sorghum populations from Bolivia were planted and evaluated for tolerance to ACC herbicides. One of the wild sorghum genotypes, Bol-71, expressed high levels of tolerance to each of the herbicides tested. It is demonstrated herein that crossing the Bol-71 wild sorghum with elite parent sorghum plant lines yields good seed set and ACC herbicide resistance in F1 hybrids. For example, seeds from cross Bol-71 and ATx623, designated KSU 06 GHATx623×174 (e.g., F1 progeny), were deposited at ATCC as described herein.
As such, one embodiment of the present invention provides a sorghum germplasm that contains altered ACC genes and proteins. In some embodiments, the present invention provides for the use of ACC herbicides in fields of hybrid sorghum crop plants to reduce the amount of monocot weed plants present in said crop field, wherein said hybrid sorghum germplasm comprises an altered ACC enzyme that confers resistance to ACC herbicides and said weed plants are ACC herbicide susceptible.
In one embodiment, the present invention provides a sorghum germplasm that confers resistance to inhibition by ACC herbicides, singly or in conjunction with other resistance traits, for example insect resistance against the spotted stem borer Chilo partellus (Girijashankar et al., 2005, Plant Cell Rep. 24:513-522, incorporated herein in its entirety). In some embodiments, for example, a sorghum hybrid whose germplasm comprises a synthetic cryl Ac gene from Bacillus thuringiensis (Bt) is introgressed into a sorghum line whose germplasm confers resistance to ACC herbicides. As well, the incorporation of ACC herbicide resistance and insect resistance is accomplished via plant transgenesis into the same sorghum hybrid. One skilled in the art will recognize the various techniques as described herein that are applicable to the incorporation of two or more resistance attributes into the same sorghum hybrid.
In one embodiment, the present invention provides ACC herbicide resistance in sorghum plants comprising, for example, an ACC germplasm designated KSU 06GH701-715bk or KSU 06GHATx623x714 deposited under ATCC accession Nos: PTA-8033 and PYA-8034, respectively, incorporated into elite sorghum varieties through plant breeding and selection, thereby providing for the development of herbicide tolerant sorghum crop hybrids that will tolerate the use of ACC inhibiting herbicides for weed control. Deployment of this herbicide tolerance trait in the aforementioned hybrids allows use of these herbicides to control monocot weeds that grow in the presence of these crops. In some embodiments, the incorporation of the ACC resistance germplasm into elite lines is via introgression, or classical breeding methods. In some embodiments, the incorporation of the ACC resistance gene into elite lines is via heterologous gene transgenesis. In some embodiments, the invention provides a sorghum hybrid, wherein at least one ancestor of the sorghum hybrid comprises an ACC resistant gene from germplasm designated KSU 06GH701-715bk or KSU 06GHATx623x714 deposited under ATCC accession Nos: PTA-8033 and PYA-8034, respectively. In some embodiments, the ACC resistant herbicide gene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714. In some embodiments, the ACC resistant herbicide gene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714 comprising a tryptophan to cysteine amino acid substitution at an amino acid position aligning with Trp2027 of the A. myosuroides Huds. ACC protein.
In some embodiments, ACC herbicide resistant germplasm is introgressed into an elite sorghum line using classic breeding techniques. Examples of classical breeding methods for sorghum can be found in, for example, Sleper and Poehlman, 2006, Breeding Field Crops, Fifth Edition, Blackwell Publishing, incorporated herein in its entirety.
In one embodiment, the ACC herbicide resistant germplasm is introgressed into a sorghum plant that provides food for human consumption. In some embodiments, the ACC herbicide resistant germplasm is introgressed into sorghum plants that provide food for livestock (e.g., poultry, cattle, swine, sheep, etc). In some embodiments, the ACC herbicide resistant germplasm is introgressed into sorghum plants that are used in industrial processes such as ethanol production. In one embodiment, the ACC herbicide resistant gene is introduced into the plant genome via transgenesis using vectors and technologies known in the art.
In some embodiments, the present invention provides an ACC resistant germplasm of a sorghum plant part of line Bol-71 and Atx623, wherein seed of said sorghum plant having been deposited under ATCC accession Nos: PTA-8033 and PYA-8034, respectively, and said sorghum plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell. In one embodiment, the present invention provides an F1 hybrid whose germplasm comprises an ACC resistance gene as described herein. In some embodiments, the F1 hybrid is a cross between two elite sorghum lines, at least one of which contains a germplasm comprising an ACC resistance gene as described herein.
In one embodiment, the present invention provides methods for controlling weeds in a field of hybrid sorghum crop plants. In some embodiments, controlling the weeds comprises applying an ACC herbicide to said field of sorghum plants, such that weed growth is inhibited but sorghum growth is not adversely affected. In some embodiments, the ACC herbicide being applied is from the aryloxyphenoxypropionate (FOP) herbicide family including, but not limited to, clodinafop-propargyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-p-ethyl, fluazifop-b-butyl, haloxyfop-ethoxyethyl, haloxyfop-etotyl, haloxyfop-R-methyl, propaquizafop, quizalofop-p-ethyl and quizalo-P-refuryl compounds. In some embodiments, the ACC herbicide being applied is from the cyclohexanediones (DIM) herbicide family including, but not limited to, alloxydim, butroxydim, clefoxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim and tralkoxydim compounds. In some embodiments, the ACC herbicide being applied comprises a combination of compounds from both FOP and DIM ACC herbicide families as disclosed herein. However, the present application is not limited to the ACC herbicide used, and a skilled artisan will appreciate that new ACC herbicides are being discovered at any given time that inhibit the ACC enzyme.
In one embodiment, the present invention provides for a sorghum hybrid (e.g., F1, F2, F3, F4, etc.) whose germplasm confers resistance to ACC herbicides and resistance to one or more additional herbicides from one or more different herbicide groups. For example, additional herbicide groups used to inhibit weed growth, include, but are not limited to, inhibitors of lipid synthesis (e.g., aryloxyphenoxypropionates, cyclohexanodeiones, benzofuranes, chloro-carbonic acids, phosphorodithioates, thiocarbamates), inhibitors of photosynthesis at photosystem II (e.g., phenyl-carbamates, pyridazinones, triazines, triazinones, triazolinones, uracils, amides, ureas, benzothiadiazinones, nitriles, phenyl-pyridines), inhibitors of photosynthesis at photosystem I (e.g., bipyridyliums), inhibitors of protoporphyrinogen oxidase (e.g., diphenylethers, N-phenylphthalimides, oxadiazoles, oxyzolidinediones, phenylpyrazoles, pyrimidindiones, thiadiazoles), inhibitors of carotenoid biosynthesis (e.g., pyridazinones, pyridinecarboxamides, isoxazolidinones, triazoles), inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (e.g., callistemones, isoxazoles, pyrazoles, triketones), inhibitors of EPSP synthase (e.g., glycines), inhibitors of glutamine synthetase (e.g., phosphinic acids), inhibitors of dihydropteroate synthase (e.g., carbamates), inhibitors of microtubule assembly (e.g., benzamides, benzoic acids, dinitroanilines, phosphoroamidates, pyridines), inhibitors of cell division (e.g., acetamides, chloroacetamides, oxyacetamides), inhibitors of cell wall synthesis (e.g., nitriles, triazolocarboxamides) and inhibitors of auxin transport (e.g., phthalamates, semicarbazones). In some embodiments, the present invention provides F1 hybrids from elite sorghum lines that comprises resistance to one or more ACC herbicides alone, or in conjunction with, herbicide resistance to one or more of the aforementioned herbicide groups.
In one embodiment, the present invention provides use of a transgene comprising a heterologous gene such as a gene encoding a mutant ACC protein for providing the selected agronomic trait of ACC herbicide resistance. In one embodiment, the transgene comprises a mutant ACC gene as found in the germplasm designated KSU 06GH701-715bk or KSU 06GHATx623x714 deposited under ATCC accession Nos: PTA-8033 and PYA-8034, respectively. In some embodiments, the transgene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714 (e.g., SEQ ID NO: 12). In some embodiments, the ACC resistant herbicide gene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714 comprising a tryptophan to cysteine amino acid substitution at an amino acid position aligning with Trp7027 of the A. myosuroides Huds. ACC protein.
Classical Breeding of Sorghum 
Field crops have been classically bred through techniques that take advantage of the plants method(s) of pollination. A plant is considered “self-pollinating” if pollen from one flower can be transmitted to the same or another flower, whereas plants are considered “cross-pollinated” if the pollen has to come from a flower on a different plant in order for pollination to occur.
Plants that are self-pollinated and selected over many generations become homozygous at most, if not all, of their gene loci, thereby producing a uniform population of true breeding progeny. A cross between two homozygous plants from differing backgrounds or two different homozygous lines will produce a uniform population of hybrid plants that will more than likely be heterozygous at a number of the gene loci. A cross of two plants that are each heterozygous at a number of gene loci will produce a generation of hybrid plants that are genetically different and are not uniform.
Sorghum plants are self-pollinating plants, but they can also be bred by cross-pollination. The development of sorghum hybrids requires the development of pollinator parents (fertility restorers) and seed parent inbreds using the cytoplasmic male sterility-fertility restorer system, the crossing of seed parents and pollinator parents, and the evaluation of the crosses. Pedigree breeding programs combine desirable traits; in the present application the desirable trait being plant resistance to ACC herbicides. This trait is put into the breeding pool from one or more lines, such that new inbred lines are created by crossing, followed by selection of plants with the desired trait, followed by more crossing, etc. New inbreds are crossed with other inbred lines (e.g., elite plant lines like those described herein).
Pedigree breeding starts with the crossing of two genotypes, such as Bol-71 and an elite sorghum line (e.g., Tx430, 00MN7645, BTx623, ATx623, Wheatland, Tx3042, OK11, QL41 and Tx643, Tx2737, Tx2783, and HP162). For example, Bol-71 wild sorghum parent was crossed to elite sorghum parent lines including Tx430, 00MN7645, BTx623 and ATx623. If the original two parents do not provide all of the desired characteristics, then other sources can be included in the breeding population. For example, if a hybrid is desired such that both ACC herbicide resistance and resistance to another herbicide group as described herein was desirous, then plants with both these attributes could be crossed using classical breeding techniques. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations, the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically, in the pedigree method, five or more generations of selfing and selection are practiced (e.g., S1, S2, S3, S4, S5, etc.).
Backcrossing is used to improve a plant line. Backcrossing transfers a specific desirable trait from one source to another that lacks the trait. This is accomplished by, for example, crossing a donor (e.g., Bol-71) to an inbred line (e.g., an elite line as described herein). The progeny of this cross is then crossed back (i.e. backcrossing) to the elite inbred line, followed by selection in the resultant progeny for the desired trait (e.g., resistance to ACC herbicides). Following five or more backcross generations with selection for the desired trait the progeny are typically heterozygous for the locus (loci) controlling the desired phenotype, but will be like the elite parent for the other genetic traits. The last backcrossing then is typically selfed in order to give a pure breeding progeny for the gene being transferred.
In current hybrid sorghum breeding programs, new parent lines are developed to be either seed-parent lines (e.g., Wheatland, Tx3042, OK11, QL41 and Tx643) or pollen-parent lines (e.g., Tx430, Tx2737, Tx2783, 00MN7645 and HP162) depending on whether or not they contain fertility restoring genes; the seed-parent lines do not have fertility restoring genes and are male-sterile in certain cytoplasms (also known as “A” line plants) and male-fertile in other cytoplasms (also known as “B” line plants), whereas the pollen-parent lines are not male sterile and do contain fertility restoring genes (also known as “R” line plants). The seed-parent lines are typically created to be cytoplasmically male sterile such that the anthers are minimal to non-existant in these plants thereby requiring cross-pollination. The seed-parent lines will only produce seed, and the cytoplasm is transmitted only through the egg. The pollen for cross pollination is furnished through the pollen-parent lines that contain the genes necessary for complete fertility restoration in the F1 hybrid, and the cross combines with the male sterile seed parent to produce a high-yielding single cross hybrid with good grain quality.
Typically, this cytoplasmic male sterility-fertility restorer system is performed for the production of hybrid seed by planting blocks of rows of male sterile (seed-parent) plants and blocks of rows of fertility restorer (pollen-parent) plants, such that the seed-parent plants are wind pollinated with pollen from the pollen-parent plant. This process produces a vigorous single-cross hybrid that is harvested and planted by the consumer. Male sterile, seed-parent plants can also be created by genetically breeding recessive male-sterile nuclear genes into a particular population, however the cytoplasmic male sterility-fertility restorer system is typically the system used for breeding hybrid sorghum. Sleper and Poehlman, 2006, Breeding Field Crops, Fifth Ed., Blackwell Publishing provides a good review of current sorghum breeding procedures and is incorporated herein in its entirety.
The present invention is not limited to the elite parent sorghum lines listed, and one skilled in the art will recognize that any elite sorghum line would be equally amenable to the compositions and methods as described herein.
Plant Transgenics
Heterologous genes intended for expression in plants are first assembled in expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements, methods of which are well known to those skilled in the art. Methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Exemplary techniques are widely described in the art (See e.g., Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., herein incorporated by reference).
In general, these vectors comprise a nucleic acid sequence encoding a heterologous gene operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.
Promoters include, but are not limited to, constitutive promoters, tissue-, organ-, and developmentally specific promoters, and inducible promoters. Examples of promoters include, but are not limited to; constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase (Chao et al., 1999, Plant Physiol 120:979-992, herein incorporated by reference); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (induced by salicylic acid and benzothiadiazole-7-carbothioic acid S-methyl ester); a heat shock promoter (U.S. Pat. No. 5,187,267, herein incorporated by reference); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422, herein incorporated by reference); and seed-specific promoters.
The expression cassettes may further comprise any sequences required for expression of mRNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
A variety of transcriptional terminators are available for use in expression of sequences using the promoters such as those disclosed herein. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (Odell et al., 1985, Nature 313:810; Rosenberg et al., 1987, Gene, 56:125; Guerineau et al., 1991, Mol. Gen. Genet. 262:141; Proudfoot, 1991, Cell, 64:671; Sanfacon et al., 1990, Genes Dev. 5:141; Mogen et al., 1990, Plant Cell, 2:1261; Munroe et al., 1990, Gene, 91:151; Ballas et al., 1989, Nucleic Acids Res. 17:7891; Joshi et al., 1987, Nucleic Acid Res., 15:9627, all of which are incorporated herein by reference).
In some embodiments, constructs for expression of the heterologous gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
In some embodiments, a construct for expression of the heterologous nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Kalderon et al., 1984, Cell 39:499; Lassner et al., 1991, Plant Molecular Biology 17:229), a plant translational consensus sequence (Joshi, 1987, Nucleic Acids Research 15:6643), an intron (Luehrsen and Walbot, 1991, Mol. Gen. Genet. 225:81), and the like, operably linked to the nucleic acid sequence encoding an heterologous gene.
In preparing the construct comprising the nucleic acid sequence encoding an heterologous gene, or encoding a sequence designed to decrease heterologous gene expression, various DNA fragments can be manipulated so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) and, as appropriate, in the desired reading frame. For example, adapters or linkers can be employed to join the DNA fragments, or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, and the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, 1982, Gene 19: 259; Bevan et al., 1983, Nature 304:184, all of which are incorporated herein by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., 1990. Nucl Acids Res. 18:1062; Spencer et al., 1990, Theor. Appl. Genet. 79:625, all of which are incorporated herein by reference), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, 1984, Mol. Cell. Biol. 4:2929, incorporated herein by reference), and the dhfr gene that confers resistance to methotrexate (Bourouis et al., 1983, EMBO J., 2:1099, incorporated herein by reference).
In some embodiments, the Ti (T-DNA) plasmid vector is adapted for use in an Agrobacterium mediated transfection process such as in U.S. Pat. No. 6,369,298 (sorghum), and U.S. Pat. Nos. 5,981,839, 6,051,757, 5,981,840, 5,824,877 and 4,940,838 all of which are incorporated by reference herein in their entireties. Construction of recombinant Ti and Ri plasmids in general follows methods typically used with more common vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include, but are not limited to, structural genes for antibiotic resistance as selection genes.
There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the “cointegrate” system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector and the non-oncogenic Ti plasmid pGV3850. The use of T-DNA as a flanking region in a construct for integration into a Ti- or Ri-plasmid has been described in EPO No. 116,718 and PCT Application Nos. WO 84/02913, 02919 and 02920; Herrera-Estrella, 1983, Nature 303:209-213; Fraley et al., 1983, Proc. Natl. Acad. Sci, USA 80:4803-4807; Horsch et al., 1984, Science 223:496-498; and DeBlock et al., 1984, EMBO J. 3:1681-1689, all of which are herein incorporated by reference.
The second system is called the “binary” system in which two plasmids are used and the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available.
In some embodiments, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No. 5,501,967 herein incorporated by reference). One of skill in the art knows that homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known. Agrobacterium tumefaciens is a common soil bacterium that causes crown gall disease by transferring some of its DNA to the plant host. The transferred DNA (T-DNA) is stably integrated into the plant genome, where its expression leads to the synthesis of plant hormones and thus to the tumorous growth of the cells. A putative macromolecular complex forms in the process of T-DNA transfer out of the bacterial cell into the plant cell.
In some embodiments, the nucleic acids as disclosed herein are utilized to construct vectors derived from plant (+) RNA viruses (e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted heterologous polynucleotide can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or another promoter. Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of which are incorporated herein by reference.
In some embodiments, a heterologous nucleic acid sequence of interest comprising a mutant ACC transgene, for example, as found in the germplasm designated KSU 06GH701-715bk or KSU 06GHATx623x714 deposited under ATCC accession Nos: PTA-8033 and PYA-8034, respectively, is introduced directly into a plant. In some embodiments, the transgene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714 (e.g., SEQ ID NO: 12). In some embodiments, the transgene is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the ACC resistant herbicide gene as found in the germplasm KSU 06GH701-715bk or KSU 06GHATx623x714 comprising a tryptophan to cysteine amino acid substitution at an amino acid position aligning with Trp7027 of the A. myosuroides Huds. ACC protein.
One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278, herein incorporated by reference).
Once a nucleic acid sequence encoding the heterologous gene is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method depends on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In some embodiments, the vector is integrated into the genome. In some embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (for example, see U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783 all of which are incorporated herein by reference in their entireties).
The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the sequences of interest into a suitable target tissue (e.g., using biolistics or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., 1990, Proc. Natl. Acad. Sci., 87:8526); Staub and Maliga, 1992, Plant Cell, 4:39, all of which are incorporated herein by reference). The presence of cloning sites between these markers allows creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga, 1993, EMBO J., 12:601). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab and Maliga, 1993, Proc. Natl. Acad. Sci., 90:913). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of RNAs encoded by the DNA molecule.
In one embodiment, vectors useful in the practice of the present invention are microinjected directly into plant cells (Crossway, 1985, Mol. Gen. Genet, 202:179). In some embodiments, the vector is transferred into the plant cell by using polyethylene glycol (Krens et al., 1982, Nature, 296:72; Crossway et al., 1986, BioTechniques, 4:320); fusion of protoplasts with other entities such as minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al., 1982, Proc. Natl. Acad. Sci., USA, 79:1859); and protoplast transformation (EP 0 292 435); direct gene transfer (Paszkowski et al., 1984, EMBO J., 3:2717; Hayashimoto et al., 1990, Plant Physiol. 93:857).
In some embodiments, the vector may also be introduced into the plant cells by electroporation. (Fromm, et al., 1985, Proc. Natl. Acad. Sci. USA 82:5824; Riggs et al., 1986, Proc. Natl. Acad. Sci. USA 83:5602). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
In addition to direct transformation, in some embodiments, the vectors comprising a nucleic acid sequence encoding a heterologous gene are transferred using Agrobacterium-mediated transformation (Hinchee et al., 1988, Biotechnology, 6:915; Ishida et al., 1996, Nature Biotechnology 14:745, all of which are herein incorporated by reference). Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for plant tumors such as crown gall and hairy root disease. In the dedifferentiated tissue characteristic of the tumors, amino acid derivatives known as opines are produced and catabolized. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. Heterologous genetic sequences (e.g., nucleic acid sequences operatively linked to a promoter of the present invention) can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens (previously described). The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell, 1987, Science, 237:1176). Species that are susceptible to infection by Agrobacterium may be transformed in vitro. Transformation methods for producing transgenic sorghum plants using Agrobacterium-mediated transformation are provided in U.S. Pat. No. 6,369,298.
In some embodiments, the vector is introduced through ballistic particle acceleration (U.S. Pat. No. 4,945,050; Casas et al., 1993, Proc. Natl. Acad. Sci. USA 90:11212, all references are incorporated herein in their entireties).
In some embodiments, after selecting for transformed plant material that can express a heterologous gene encoding a heterologous protein or variant thereof, whole plants are regenerated. Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York, (1983); Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, (1984) and Vol. III, (1986), incorporated herein by reference in their entireties. It is known that many plants can be regenerated from cultured cells or tissues including, but not limited to, all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, and monocots (e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
EXAMPLES
Example 1
Herbicide Resistance in Wild Sorghum Genotype
Seeds of 83 wild sorghum populations from Bolivia were planted in a greenhouse for comparison with Tx2783, an herbicide susceptible elite sorghum genotype. Wild sorghum genotypes were planted in flats containing MetroMix 360 potting soil (Sun Gro) and greenhouse grown. Tx2783 was planted with the wild accessions in each flat for comparison. In one selection using clethodim, plants were sprayed with 0.09 lb ai clethodim acre−1 at 18 days after planting. Tx2783 and many of the wild sorghums died, but the herbicide tolerant entries were transplanted to pots for seed increase. In one selection using fluoazifop-P, flats with Tx2783 and wild accessions were sprayed with a rate of 0.12 lb ai fluazifop-P acre−1 at 18 days after planting and with 0.36 lb ai fluazifop-P acre−1 at 32 days after planting. Tx2783 and many of the wild sorghums died, but the herbicide tolerant entries were transplanted to pots for seed increase. In a third experiment, plants were sprayed with a rate of 0.05 lb ai quizalofop acre−1 at 18 days after planting and with 0.11 lb ai quizalofop acre−1 at 32 days after planting. Tx2783 and many of the wild sorghums died, but the herbicide tolerant entries were transplanted to pots for seed increase. One of the wild sorghum genotypes, Bol-71, expressed high levels of tolerance to each of the herbicides.
Example 2
Crosses of Bol-71 Wild Sorghum Genotype with Elite Sorghum Lines and Inheritance Determination
Bol-71 was crossed with elite sorghum parent lines including Tx430, OOMN7645, BTx623, and ATx623. Seed set was excellent in every cross indicating that the wild genotype was sexually compatible with cultivated sorghum and could be used in a plant breeding program to produce herbicide tolerant sorghum varieties.
The mode of inheritance of herbicide tolerance was determined by planting seeds of Bol-71, F1 generation of cross ATx623×Bol-71, and Pioneer 84G62 (herbicide susceptible control) in flats containing MetroMix 360 potting soil in a greenhouse using a randomized complete block design (n=3). The plants were sprayed with 0.045 lb ai fluazifop-P acre−1 at 14 days after planting. Pioneer 84G62 died between 12-16 days after spraying. The ATx623×Bol-71 and Bol-71 genotypes showed no herbicide damage, indicating that the herbicide tolerance trait was transmitted to cultivated sorghum and that herbicide tolerance was at least partially dominant in F1 hybrids.
Example 3
Gene Sequencing for ACC Resistance Gene
Gene sequencing efforts were initiated to determine if a genetic mutation might explain the herbicide tolerance phenotype. DNA was extracted from herbicide tolerant genotypes Bol-71 and R91 and herbicide susceptible genotypes Bol-36, ATx623, and Tx430. The polymerase chain reaction (PCR) using primers described by Délye and Michel (Weed Research, 2005, 45: 323-330; incorporated herein in its entirety) were used to amplify regions of the ACC gene associated with expression of herbicide tolerance. DNA sequencing (Kansas State University DNA sequencing facility) of resultant PCR products from herbicide tolerant and susceptible sorghum genotypes revealed that the susceptible genotypes contained the wild type sequence for the ACC gene as reported for sorghum (The Institute for Genomic Research (TIGR) Plant Transcript Assemblies database sequence designated TA3768_4558; incorporated herein in its entirety, see also table 1 below) and other cereal crops species; however, the herbicide tolerant genotypes contained a genetic mutation of TGG to TGC that results in a tryptophan to cysteine amino acid conversion at an amino acid position aligning with Trp2027 of the A. myosuroides Huds. ACC protein (SEQ ID NO: 12) in the enzyme (FIG. 1; see Délye and Michel, Weed Research 2005 for amino acid numbering). This mutation is similar to the Trp2027Cys amino acid conversion in the ACC enzyme described in herbicide tolerant blackgrass (Alopecurus myosuroides Huds.) by Délye et al. (Plant Physiology 2005). The wild type A. myosuroides Huds. ACC protein (see Délye and Michel, Weed Research, 2005, 45:323-330; incorporated herein in its entirety) is represented by SEQ ID NO: 13. Blackgrass is a weed species and the only place in nature where this mutation has been reported, and the mutation has not been reported in sorghum or other crop species, or described as a target for developing herbicide tolerant crops.







TABLE 1





Sorghum ACC Carboxylase















Portion of the Wild Type Sorghum ACC Carboxylase


Gene Sequence


(SEQ ID NO: 1)


TGGCAGAGCAAANCTTGGAGGAATTCCTGTGGGTGTCATAGCTGTGGAGA


 


CACAGACCATGATGCAGCTTGTCCCTGCTGATCCAGGTCAGCTTGATTCC


 


CATGAGCGATCCGTTCCTCGGGCTGGACAAGTGTGGTTCCCAGATTCTGC


 


AACCAAGACAGCTCAGGCATTATTAGACTTCAACCGTGAAGGATTGCCTC


 


TGTTTATCCTGGCTAACTGGAGAGGTTT


                 ***


               [Trp2031]


 


CTCTGGTGGACAGAGAGATCTCTTTGAAGGAATTCTTCAGGCTGGGTCAA


 


CAATTGTCGAGAACCTTAGGACATATAATCAGCCTGCGTTTGTCTACATT


 


CCTATGGCTGGAGAGCTTCGTGGAGGAGCTTGGGTTGTGGTCGATAGCAA


 


AATAAATCCAGACCGCATTGAGTGTTATGCTGAGAGGACTGCCAAAGGTA


 


ATGTTCTCGAACCTCAAGGGTTAATTGAAATCAAGTTCAGGTCAGAGGAA


 


CTCCAAGACTGTATGGGTAGGCTTGACCCCGAGTTGATAAATCTGAAAGC


 


AAAACTCCAAGATGTAAAGCATGGAAATGGAAGTCTACCAGACATAGAAT


 


CCCTTCAGAAGAGTATAGAAGCACGTACGAAACAGTTGCTGCCTTTATAT


 


ACCCAGATTGCAATACGGTTTGCTGAATTGCATGATACTTCCCTAAGAAT


 


GGCAGCTAAAGGCGTGATTAAGAAAGTTGTAGACTGGGAAGAATCACGCT


 


CTTTCTTCTATAAAAGGCTACGGAGAAGGATCTCTGAAGATGTTCTTGCA


 


AAAGAAATAAGACATATAGTCGGTGACAACTTCACTCACCAATCAGCAAT


 


GGAGCTCATCAAGGAATGGTACCTGGCTTCTCCAGCCACAGCAGGAAGCA


 


CTGGATGGGATGACGATGATGCATTTGTTGCCTGGAAGGACAGTCCTGAA


 


AACTACAATGGATATATCCAAGAGCTAAGGGCTCAAAAAGTGTCTCAGTC


 


GCTCTCTGATCTCACTGACTCCAGTTCAGATCTACAAGCATTCTCGCAGG


 


GTCTTTCTACGCTATTAGATAAGATGGATCCCTCTCAAAGAGCGAAGTTT


 


GTTCAGGAAGTCAAGAAGGTCCTTGGTTGATGATATGATACCAACACATC


 


CAACACTATGTGCATGCTACATGTTTTTGTTCAAGTACATACATAGAAGG


 


ATATTGCTTGGCCTCGTTTGCTTGGCCGTTTGATCATGTCTGATCTAAGT


 


CGACCATTATTTGTTGAAACTTCCTTTTTGGACCTGGTGCTATGGTTGAT


 


GAATGTATATTGGACGTGTGCGACTGTGCGTTCTGCCAGGTGTAAGCTCA


 


AATGTTTAGACAGACCGAGTTATGGTTAGGAAGAGCACGAGTGAACATGT


 


TCTGGTTTTGCAGTGGTTCAGGAAGGCAGAAAGTTGTTTCACTGTAGTTC


 


TGAGATGTACTACCAGCGGCCCCCTGCTGTAATTTTAGGGTGTATAATGC


 


GGATACTAGTAAAACAATTGAGTGGTTCATTAAATTTTGAACTCGAATAA


 


TGTTTTTCTAGGCATATGTACCGTACCTCTACGTGAAATAAATGCTGTTG


 


AAATAGCATTCGACACCAGAATATATGTACCTTACCTAAGAGCTAAGTAT


 


TATAATACAACAAGTTGCTGGCCGTAAGATTTCTTT


 


Portion of the Sorghum ACC Carboxylase Gene


Sequence Encoding for Trp2031Cys


(SEQ ID NO: 12)


TGGCAGAGCAAANCTTGGAGGAATTCCTGTGGGTGTCATAGCTGTGGAGA


 


CACAGACCATGATGCAGCTTGTCCCTGCTGATCCAGGTCAGCTTGATTCC


 


CATGAGCGATCCGTTCCTCGGGCTGGACAAGTGTGGTTCCCAGATTCTGC


 


AACCAAGACAGCTCAGGCATTATTAGACTTCAACCGTGAAGGATTGCCTC


 


TGTTTATCCTGGCTAACTGCAGAGGTTT


                 ***


              [Trp2031Cys]


 


CTCTGGTGGACAGAGAGATCTCTTTGAAGGAATTCTTCAGGCTGGGTCAA


 


CAATTGTCGAGAACCTTAGGACATATAATCAGCCTGCGTTTGTCTACATT


 


CCTATGGCTGGAGAGCTTCGTGGAGGAGCTTGGGTTGTGGTCGATAGCAA


 


AATAAATCCAGACCGCATTGAGTGTTATGCTGAGAGGACTGCCAAAGGTA


 


ATGTTCTCGAACCTCAAGGGTTAATTGAAATCAAGTTCAGGTCAGAGGAA


 


CTCCAAGACTGTATGGGTAGGCTTGACCCCGAGTTGATAAATCTGAAAGC


 


AAAACTCCAAGATGTAAAGCATGGAAATGGAAGTCTACCAGACATAGAAT


 


CCCTTCAGAAGAGTATAGAAGCACGTACGAAACAGTTGCTGCCTTTATAT


 


ACCCAGATTGCAATACGGTTTGCTGAATTGCATGATACTTCCCTAAGAAT


 


GGCAGCTAAAGGCGTGATTAAGAAAGTTGTAGACTGGGAAGAATCACGCT


 


CTTTCTTCTATAAAAGGCTACGGAGAAGGATCTCTGAAGATGTTCTTGCA


 


AAAGAAATAAGACATATAGTCGGTGACAACTTCACTCACCAATCAGCAAT


 


GGAGCTCATCAAGGAATGGTACCTGGCTTCTCCAGCCACAGCAGGAAGCA


 


CTGGATGGGATGACGATGATGCATTTGTTGCCTGGAAGGACAGTCCTGAA


 


AACTACAATGGATATATCCAAGAGCTAAGGGCTCAAAAAGTGTCTCAGTC


 


GCTCTCTGATCTCACTGACTCCAGTTCAGATCTACAAGCATTCTCGCAGG


 


GTCTTTCTACGCTATTAGATAAGATGGATCCCTCTCAAAGAGCGAAGTTT


 


GTTCAGGAAGTCAAGAAGGTCCTTGGTTGATGATATGATACCAACACATC


 


CAACACTATGTGCATGCTACATGTTTTTGTTCAAGTACATACATAGAAGG


 


ATATTGCTTGGCCTCGTTTGCTTGGCCGTTTGATCATGTCTGATCTAAGT


 


CGACCATTATTTGTTGAAACTTCCTTTTTGGACCTGGTGCTATGGTTGAT


 


GAATGTATATTGGACGTGTGCGACTGTGCGTTCTGCCAGGTGTAAGCTCA


 


AATGTTTAGACAGACCGAGTTATGGTTAGGAAGAGCACGAGTGAACATGT


 


TCTGGTTTTGCAGTGGTTCAGGAAGGCAGAAAGTTGTTTCACTGTAGTTC


 


TGAGATGTACTACCAGCGGCCCCCTGCTGTAATTTTAGGGTGTATAATGC


 


GGATACTAGTAAAACAATTGAGTGGTTCATTAAATTTTGAACTCGAATAA


 


TGTTTTTCTAGGCATATGTACCGTACCTCTACGTGAAATAAATGCTGTTG


 


AAATAGCATTCGACACCAGAATATATGTACCTTACCTAAGAGCTAAGTAT


 


TATAATACAACAAGTTGCTGGCCGTAAGATTTCTTT









All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

## Claims

1. A seed producing the germplasm of sorghum hybrid variety KSU 06 GHATx623x714, wherein representative seeds have been deposited under ATCC No. PYA-8034, and wherein:
(i) said germplasm comprises a sorghum acetyl-CoA carboxylase gene comprising a 1664-base portion having a nucleotide sequence represented by SEQ ID NO: 12,
(ii) said sorghum acetyl-CoA carboxylase gene encodes a sorghum acetyl-CoA protein having a tryptophan to cysteine amino acid substitution at an amino acid position aligning with Trp2027 of SEQ ID NO: 13, and
(iii) the expression of said sorghum acetyl-CoA carboxylase gene confers to said sorghum hybrid grown from said seed resistance to inhibition by one or more aryloxyphenoxypropionate acetyl-CoA carboxylase herbicides at levels of said one or more aryloxyphenoxypropionate herbicides that would normally inhibit the growth of a sorghum hybrid.

2. A method of controlling weeds in the vicinity of a cultivated sorghum hybrid, said method comprising:
a) providing one or more acetyl-CoA carboxylase herbicides,
b) applying said one or more acetyl-CoA carboxylase herbicides to a field comprising a sorghum hybrid plant grown from the seed of claim 1, and
c) controlling weeds in the vicinity of said cultivated sorghum hybrid such that weed growth is adversely affected by the application of said one or more herbicides and growth of said cultivated sorghum hybrid is not adversely affected.

3. A method of producing an herbicide resistant sorghum plant, said method comprising crossing a sorghum hybrid plant grown from the seed of claim 1 with a different sorghum plant and selecting a progeny plant comprising SEQ ID NO: 12.