WO2014113846A1

WO2014113846A1 - Herbicide tolerant barley

Info

Publication number: WO2014113846A1
Application number: PCT/AU2014/000060
Authority: WO
Inventors: Jason Konrad EGLINTON; Suong CU; Shannan LARWOOD; Gurjeet GILL; Ben FLEET; Christopher Preston; Peter Boutsalis; Jenna MALONE
Original assignee: Adelaide Research & Innovation Pty Ltd
Priority date: 2013-01-25
Filing date: 2014-01-24
Publication date: 2014-07-31
Also published as: AU2014210372B2; AU2014210372A1; AR094594A1

Abstract

Herbicide resistant barley plants and compositions and methods for producing herbicide resistant barley hybrids, in particular barley plants with mutated acetyl-CoA carboxylase (ACCase) genes that are resistant to ACCase inhibiting herbicides, such as barley varieties BOZO and HT107.

Description

HE B!CIDE TOLERANT BARLEY

Field of tt\& Invention The present invention provides for compositions and methods for producing commercial barley plants thai are resistant to herbicides, in particular, the present invention provides for barley plants, plant tissue and plant seed that contain acetyl-CoA carboxylase (ACC) genes and proteins that confer resistance to inhibition by herbicides that normally inhibit the activity of the ACC protein.

Background

Barley (Hordeum vuigere) is a major cereal grain and is a member of the grass family. Barley is used in malt production, brewing and for the production oi cereal foods for human and animal consumption and is generally ranked in the top five cereal crops globally in terms of quantity produced and area under cultivation.

The control of weeds is a significant factor in the economic production of barley. The use of chemical herbicides to control weeds is popular and more desirable in most circumstances compared to mechanical cultivation, which is generally more expensive and can result in damage to soil structure and erosion .

Of particular interest to farmers is the use of herbicides with greater potency, broad weed spectrum effectiveness and rapid soil degradation. One such class of broad spectrum herbicides, are those compounds that inhibit the activity of the acetyl-CoA carboxylase (ACC) enzyme in a plant. These are known as 'Group A^! or 'G o p T herbicides and include compounds within the following classes; aryloxyphenoxyproplonates (FOP); cyciohexanediortes (DIM); and phenyipyrazoiins.

While FGP and DIM herbicides are generally the first line of choice for removing grass weeds in broadleaf (dicot) crops, most of them cannot be used selectively in cereals including barley. Therefore, grass weeds are sprayed with non-selective herbicides such as g!yphosate before seeding barley but grass weeds that emerge later in the crop cannot be controlled selectively at present.

There is a need in the art for improved barley varieties and methods for improving the production of barley.

The present invention overcomes some or all of the shortcomings of the prior art.

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(Rule 91 ) ISA/AU Symmafy of the invention

The present invention provides for barley plants and barley hybrids and methods for producing barley hybrids that are resistant to herbicides, in particular the present invention provides for bariey plants and hybrids, plant tissue and plant seed that contain acety!-CoA carboxylase (ACC) genes and proteins that confer resistance to Inhibition by herbicides that normally inhibit the activity of the AGC protein.

Cultivated commercial bariey is susceptible to many ACC inhibiting herbicides that target monocot or grassy weed species. However, as described herein a barley genotype was developed that exhibits tolerance to ACC inhibiting herbicides. Genetic analysis has Identified genetic differences within germplasm that results in a ACC herbicide resistant phenoiype.

In one embodiment, the present invention provides for one or more barley plants whose germplasm comprises a mutation that renders the plani tolerant to ACC herbicides. Moreover, in further embodiments the invention relates to the offspring {e.g., Fl, 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 barley hybrids whose germplasm contains a mutation, such that the phenoiype of the plants is ACC herbicide resistant. In some embodiments, said offspring {e.g., Fl. F2, F3, etc.) are the result of a cross between elite bariey lines, at least one of which contains a germplasm comprising a mutation that renders the plant tolerant to ACC herbicides.

In a first aspect, the Invention is a barley plant, wherein said barley plant confers resistance to inhibition by one or more acety!-CcA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant.

Preferably, the barley plant is a commercial cultivar.

Preferably, the acetyl-CoA carboxylase inhibiting herbicide is selected from the group consisting of; an aryloxyphenoxypropionate herbicide; and a cyciohexanedione herbicide. Mo e preferably, the aryloxyphenoxypropionate herbicide is selected from the group consisting of; clodinafop-propargyi; diciofop-methyl; quiza!ofop-p-ethy!; fenoxyprop-p- ethyl; haloxyfop; fluazifop; and propaquizafop. More preferably, the cyciohexanedione herbicide is selected from the group consisting of; c!eihod!m; tralkoxydim; sethoxydim; tepra!oxydim; and butroxydim.

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(Rule 91) ISA/AU in one preferred embodiment, the barley plant comprises an acetyl-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: iie ₍j₃₃Leu; iie Vai; and !ie i_93SLeu and He zosaVai. For example, the !soieucine at position 1933 (see sequence listirsg for sequence references) has beers substituted with a Leucine at the same position.

Preferably, the barley plant comprises an acetyi-GoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the carboxyl transferase domain comprises anyone of the amino acids or polypeptides selected from the group consisting of;

* i93sLeu and zoea ai;

« SEQ ID Mo 1 ;

* SEQ ID No 2;

SEQ ID No 1 and SEQ ID No 2;

« SEQ ID No 3; « SEQ ID No 4; and

SEQ ID No 8.

Preferably, the barley plant comprises an acety!-CcA carboxylase gene further comprising 3 polynucleotide that corresponds to a carboxyl transferase domain; and wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises anyone of the nucleic acids and polynucleotides selected from the group consisting of;

* sw TA;

♦ ₅₂₇₇GTT;

SEQ ID No 5

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(Rule 91 ) ISA/AU SEO, !D No 8;

SEQ ID No 5 and SEQ ID No 8; and SEQ ID No 7.

In one embodiment, the polynucleotide is introduced into the germpiasm of the barley plant by hybridisation. Preferably, the polynLideotsde Is introduced into the germpiasm of the bartey plant by introgression. Methods for these techniques are widely available in the art. See also Principles of Plant Breeding, 2nd ed By R. W. Allard. John Wiley & Sons, Mew York. 1999. Alternatively, the polynucleotide is introduced into the germpiasm of the barley plant by recombinant DMA engineering techniques. Methods for these techniques are widely available in the art. See also in Vitro Plant Breeding S. Thirugrtanakumar, K. Manivannan, M, Prakash, R. Narasimman and Y. Anitha. Vasiine, Agrobios, 2009 in another preferred embodiment, the barley plant is not genetically modified.

in a second aspect, the invention is a seed of a barley plant as described herein.

In a third aspect, the Invention is a germpiasm of a barley plant as described herein.

In a fourth aspect, the invention is a method of producing a barley hybrid with increased resistance to inhibition by one or more aceiyi-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a arley plant, said method comprising; selecting a first barley plant;

selecting a second barley plant;

crossing the first barley plant with the second barley plant to produce hybrid offspring:

selecting a hybrid from said hybrid offspring with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides thereby to produce the hybrid: and

wherein the second barley plant is a barley plant as described herein.

in a fifth aspect, the invention is a method of producing a barley hybrid with increased resistance to inhibition by one or more acetyl-GoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant, said method comprising; selecting a first barley piant;

isolating cells from the first barley plant;

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(Rule 91) ISA/AU incorporating a foreign polynucleotide into the carboxy! transferase domain of the genome of the said cells of the first barley plant using recombinant DNA engineering techniques;

growing offspring from the said cells;

selecting offspring with increased resistance to inhibition by one or more acetyl-

CoA carboxylase Inhibiting herbicides thereby to produce the barley hybrid; and wherein the foreign polynucleotide is selected from polynucleotides that correspond to a carboxy! transferase domain of a second barley plant:

wherein the second barley plant is a barley plant as described herein; and wherein the foreign polynucleotide is selected from the group consisting of;

(a) _M97TTA;

(c) e TTA and s^GTT;

(d) SEQ ID No 5

(&) SEQ ID No 6;

(f) SEQ ID No 5 and SEQ !D No 6; and

(g) SEQ ID No 7,

Preferably, in the fourth and fifth aspects described above, the first barley plant is selected from the group consisting of AC Metcalfe, Bass, Baudin, Bu!oke, CDC Cope!and, CDC Reserve, Commander, Fairview, Fathom, Flagship, Fleet, Gairdner, Henley, Hindmarsh, Keel, Navigator, Oxford, Propino, Scope, Sebastian, Skipper, SouthemStar, Quench, Vlamingh, V Admiral, Westminster, WI4593 and Wimmera. Preferably, In the fourth and fifth aspects described above, the second barley plant is selected from the group consisting of; BOZO; and HT017.

In a sixth aspect, the Invention is a barley hybrid produced by the methods herein described.

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(Rule 91 ) ISA/AU in one embodiment, the hybrid comprises an acetyi-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: ile^ssLeu; !ieaei_&Va!; and ile _!83SLeu and liejooaVai.

Preferably, the hybrid comprises a acetyl-GoA carboxylase gene, further comprising a polynucleotide that corresponds to a carboxyi transferase domain; and

wherein the carboxyl transferase domain comprises anyone of the amino acids or polypeptides selected from the group consisting of;

* is₃sLeLi;

* SEQ ID No l ; SEQ ID No 2;

SEQ ID No 1 and SEQ ID No 2;

* SEQ ID No 3;

* SEQ ID No 4; and SEQ ID No. 8.

Prefsrab!y, the hybrid comprises an acetyi-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyi transferase domain; and

wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises nucleic acids selected from the group consisting of;

* swTTA;

5_?77GTT; /ΤΤΑ and _6S77G^'TT;

SEQ ID No 5;

SEQ ID No 6;

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(Rule 91 ) ISA/AU SEQ ID No 5 and SEQ ID No 6; and

SEQ ID No 7.

Preferably, the barley hybrid is a commercial cu!iivar. That is, it is adapted for us in the commercial production of barley.

In a seventh aspect, the Invention is a seed of a bariey hybrid as herein described.

in an eighth aspect, the invention is germp!asm of s barley hybrid as herein described.

!n a ninth aspect, the invention is a method of producing bariey, the method comprising;

(1 ) selecting a bariey plant as described herein;

(2) growing the said barley plant;

(3) harvesting bariey from the barley plant; and

wherein the barley plant has increased resistance to inhibition by one or more acety!-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a bariey plant.

In a tenth aspect, the invention is a method of producing barley, the method comprising;

(1 ) selecting a barley hybrid as described herein;

{2} growing the said bariey hybrid;

(3) harvesting barley from the barley hybrid; and

wherein the hybrid has increased resistance to Inhibition by one or more acetyi-CoA carboxylase inhibiting herbicides at Ievels that would normally inhibit the growth of a barley hybrid.

Preferably, the methods of the ninth and tenth aspects of the invention improve weed control compared to weed control normally observed for producing bariey.

Preferabiy, the methods of the ninth and tenth aspeds of the invention decrease contamination with weed species compared to contamination normally observed for producing barley.

in an eleventh aspect, the Invention provides a method of controlling weeds in the vicinity of a bariey plant as described herein, comprising:

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( ule 91 ) ISA/AU (a) providing one or more acety!-CoA carboxylase inhibiting herbicides

(b) applying said one or more acetyl-GoA carboxylase inhibiting herbicides to a field comprising the barley plant as described herein: and

(c) controlling weeds in the vicinity of said barley plant such that weed growth Is adversely affected by the application of said one or more herbicides and growth of said bariey plant is not adversely affected.

In a twelfth aspect, the invention provides a method of controlling weeds in the vicinity of a bariey hybrid as described herein, comprising:

(a) providing one or more acety!-GoA carboxylase inhibiting herbicides

(b) applying said one or more acety!-CoA carboxylase inhibiting herbicides to a field comprising the barley hybrid of as described herein; and

(c) controlling weeds in the vicinity of said barley hybrid such that weed growth is adversely affected by the application of said one or more herbicides and growth of said bariey hybrid is not adversely affected.

Preferably, in the methods of the eleventh and twelfth aspects of the invention, the acetyl- CoA carboxylase inhibiting herbicide is selected from the group consisting of; an aryloxyphenoxypropionate herbicide and a cyciohexanedione herbicide. Preferably, the aryioxyphertoxypropionate herbicide is selected from the group consisting of; e!odinafop- propargy!; dic!ofop-methyl; fenoxyprop-p-ethy!; quizalofop-p-sthyl; haloxyfop; fluazifop; and propaquizafop. Preferably, the cyciohexanedione herbicide is selected from the group consisting of; ctethodim; sethoxydim; tepraloxydim; tepraloxydim; tralkoxydim; and butroxydim.

Preferably, the phenylpyrazo!in herbicide is pinoxaden

Preferably, the one or more acetyi-CoA carboxylase inhibiting herbicides is applied at a rate selected from the group consisting of: between 5m!/ha and l OOOml/ha; between l Omi/ha and 50Qml/ha; between 50m! 'ha and 400ml/ha; between 10Gmi/ha and 400m!/ha; between 200rni/ha and 400mi/ha.

In a thirteenth aspect, the invention is a barley plant or barley hybrid as described herein, wherein said barley plant or said bariey hybrid confers resistance to inhibition by one or more acetyl-GoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a bariey plant, wherein said levels are selected from the group consisting of the following herbicide application rates: between 5mi/h3 and 1000mi/ha; between 10ml/ha

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(Rule 91 ) ISA/AU and 500ml/ha; between 50ml/ha and 4GQml/ha; between 1 G0rnl/ha and 400ml/ha; between 200ml/ha and 400ml ha; beiow 1000m!/ha; below SOQmi/ha; below 400mi ha; below 30Gm!/ha; below 200rol/ha: beiow 100mi/ha; beiow 50mi/ha; beiow 40ml/ha; below 30m!/ a; beiow 20mi/ha; and beiow 10ml/ha.

in one preferred embodiment of the invention, the carboxyi transferase domain of the barley plant or hybrid as described herein does nol have 100% sequence homology to the following amino acid sequence : ,s₃cCys Gki A.sn Leu His G!y Ser es_.

In one preferred embodiment of the invention, the barley plant or barley hybrid as described herein is sensitive to acetolactate synthase enzyme inhibitor herbicides. Preferably, the barley plant is sensitive to acetolactate synthase enzyme inhibitors herbicides selected from the group consisting of: Imidazolines and sulfonylureas.

Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description.

Brief Description of the Drawings

Figure 1 : This figure presents the graphical representation of the effects of four ACGase- inhibitlng FOP herbicides on the grain yield of barley mutant BOZO and cuitivar Fleet as discussed in Example 2. Treatments with the same letter above the bar are not statistically significant based on the least significant difference at P=0.CS.

Figure 2: This figure presents the photographies! data of the effects of four ACCase- inhibiting FOP herbicides on the grain yield of BOZO and cuitivar Fleet as discussed in Example 2. BOZO is shown to the left of each photo and cuitivar Fleet is shown in the right of each photo. Herbicide was applied perpendicular to sowing direction (left to right). A - 5^th of August 2011 (10 DAA), B - 19^th of August 2011 (23 DAA), G - 1^3f of September 2011 (37 DAA), and O - 11^!!l of November 2011 (108 DAA),

Figure 3: This figure presents the graphical representation of the effects of three ACGase- inhibiting DIM herbicides on the grain yield of barley mutant BOZO and cuitivar Fleet as discussed in Example 2. Treatments with the same letter above the bar are not statistically significant based on the least significant difference at P~Q.05.

Figure 4: This figure presents HT017 three weeks after treatment with Fusi!ade in comparison to sensitive genotypes.

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(Rule 91 ) ISA/AU Detaiied Description of the invention

General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes aii such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or Indicated in the specification, individiiaiiy or collectively and any and all combinations or an two or more of the steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are dearly within the scope of the invention as described herein.

The invention described herein may include one or more ranges of values (e.g. size, concentration etc). A range of values will be understood to include ali values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. Inclusion does not constitute an admission is made that any of the references constitute prior art or are part of the common general knowledge of those working In the field to which this invention relates.

The disclosures of aii publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein does not constitute an admission that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations, such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer, or group of integers, but not the exclusion of any other integers or group of Integers. It is also noted that in this disclosure, and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in US Patent law; e.g., they can mean "includes", "included", "including", and the like.

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(Rule 91) ISA/AU The term "barley plant" refers to the species Hordeum vulgars in its total genetic variation and its many varieties aiid to related species within the Hordeum genus. For example, the term includes the following varieties: AC Metcalfe, Bass, Baudlrt, Buioke, CDC Copeland. CDC Reserve, Commander, Fairview. Fathom, Flagship, Fleet, Gairdner, Henley, 5 Hindmars , Keel, Navigator, Oxford, Propirto, Scope, Sebastian, Skipper, SouihernStar, Quench, Vlamingh, VT Admiral, Westminster, WI4593 and Wimmera.

The terms "increased resistance to herbicides'', "herbicide resistant barley" and "resistance to inhibition by one or more acely!-CoA carboxylase inhibiting herbicides" mean that the plant is not as adversely affected by the herbicide as its naturally occurring

10 counterpart or wild type. The term includes tolerance to herbicides and reduced sensitivity. Preferably, the herbicide does not kill the herbicide resistant barley. In one embodiment, the herbicide only kiiis a small percentage of herbicide resistant barley plants in a crop selected from a group of percentage ranges consisting of: less than 1 % of the barley crop; less than 2%; less than 5%; less than 10%; less than 20%; less than

15 30%; less than 40%: and less than 50%. Preferably, the herbicide does not reduce the growth rate of the herbicide resistant barley, in one embodiment, the herbicide reduces the growth rate of the herbicide resistant barley by a percentage range selected from the group consisting of. less than a 1% reduction in growth rate; less than 2%. !ess than 5%; less than 10%; less than 20%; less than 30%; less than 40%; and less than 50%.

20 Preferably, the herbicide does not reduce the yield rate of the herbicide resistant barley, in one embodiment, the herbicide only reduces the yield rate of the herbicide resistant barley crop by a percentage range selected from the group consisting of: less than 1 % of the herbicide resistant barley crop: less than 2%; less than 5%; less than 0%; less than 20%; less than 30%: less than 40%; and less than 50%.

25 The term "acetyS-GoA carboxylase inhibiting herbicides" are those herbicides that inhibit acetyi-GoA carboxylase (ACC) enzyme in a plant. These are known as 'Group A' or 'Group 1 ^* herbicides and include compounds within the following classes; ary!oxyphenoxypropionates (FOP); cyclohexanediones (DIM); and phenylpyrazoiins.

The term "carboxyl transferase domain" refers to the domain on a polypeptide or protein 30 which carries out the reaction of transcarboxyiation from biotin to an acceptor molecule.

There are two recognised types of carboxyl transferase. One of them uses acy!-CoA and the other uses 2-αχα acid as the acceptor molecule of carbon dioxide.

The invention further provides a seed of the non-naiurally occurring barley plant which has increased resistance to herbicides.

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(Rule 91) ISA/AU While nor wishing to be bound by theory, it appears thai the increased herbicide resistance developed by the present inventors results from changes to the physiolog and/or biochemistry of the p!ant and thus interferes with the norma! action of the herbicide. This may involve altered absorption and/or translocation of ihe herbicide or altered ability of metabolic enzymes to bind the herbicide. Thus the invention extends to the altered elements in the plant that confer herbicide resistance including altered gene(s) and/or proteln(s) including enzymes, which, genes and proteins are altered compared to their counterparts in herbicide sensitive barley plants. Preferably said altered genes and/or proteins are in isolated or substantially purified form.

The present invention will now be described with reference to the following non-limiting Examples. The description of the Examples is in no way limiting on the preceding paragraphs of this specification, but is provided for exemplification of the methods and compositions of the invention.

Examptes

It will be apparent to persons skilled in the materials and biological arts that numerous enhancements and modifications can be made to the above described processes without departing from the basic inventive concepts. All such modifications and enhancements are considered to be within the scope of the present invention, the nature of which is to be determined from the foregoing description and the appended claims. Furthermore, the following Examples are provided for illustrative purposes only, and are not intended to limit the scope of the processes or compositions of the invention,

EXAMPLE 1

The inventors developed a barley variety, named 'BOZO', whose phenotype is a reduced sensitivity to ACC herbicides. Genetic analysis has identified genetic differences within the germp!asm of BOZO (compared barley plants that are ordinarily sensitive to ACC herbicides) that results in an ACC herbicide resistant phenotype. The genotype of BOZO was compared to three different barley varieties.

Plant materials consisted of four different barley lines; Flagship, Alexis. BOZO and Clipper. Genomic DMA was extracted from a single cotyledon of 6-day old seedlings and used as the template for polymerase chain reactions (PCR). Three sets of PGR primers were designed based on the published sequence of aceiyi-CoA carboxylase rnRNA from black grass ASopecurus myosuroides (GenBank: AJ310787) to amplify approximately 7.5 kb of carboxyi-transferase (CT) domain encompassing the regions known to be involved in sensitivity to ACCase herbicides (De^'lye C and Michel S 2005 "Universal" primers for

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(Rllle 91 ) ISA/AU PCR-sequencirig of grass ch!oroplastic aeetyl-CoA carboxylase domains involved in resistance to herbicides- Weed Res 45: 323-330). PGR amplification was carried out using standard conditions. All primer sets yielded single amplification products of expected sizes which were then purified and sequenced in both 5^' and 3^'directions using either the forward or reverse primers as used for the Initial PGR. Sequencing reactions were carried aut at the Australian Genome Research Facility, Waits Campus. University of Adelaide, SA. Sequences were then compiled and aligned against the black grass sequence AJ310767 using SioEdit sequence alignment editor (hltp:// ¾'w.mbio.ncsu.edu/ ioediij''bloedit.html) and assessed for polymorphisms, A single nucleotide polymorphism (SNP) was identified in BOZO compared to the reference barley varieties Alexis, Flagship and Clipper. The SNP is at base position 5497 transitioning from A to T which results in an amino acid change from isoleucine to leucine (Tab!el ).

lab!e 1. Summary of sequencing results showing the codons of interest and the sequence found at that position in each of the four varieties.

The region of DNA in BOZO known in other plant varieties to be Involved In sensitivity to ACOase herbicides in the carboxyl transferase domain, was sequenced and is presented in SEQ ID 7.

EXAMPLE 2

The extent of BOZO's resistance to ACCase herbicides was characterized in a field trial.

The aim of the trial was to investigate tolerance of BOZO to four AGCase-inhibiting FOP herbicides, three AGCase-inhibiting DIM herbicides and one ALS-inhibiting imidazolinone herbicide under field conditions.

Met ods

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(Rule 91 ) ISA/AU A field trial was conducted at Roseworthy Campus in the iower norih region of the South Australia in 2011. The Roseworthy site has a Red-brown earth soil type and a Mediterranean type climate with an average annual rainfall of 440mrn. The site was previously crapped to field peas and had a low weed pressure and good soil fertility The trial was established In a replicated spilt plot design with BOZO barley and Fleet cu!iivar as the main plots and various herbicide treatments as split plots. Each plot was 1.5m wide and 5m long and each treatment was replicated 3 times. Barley strips were sown with a no-tiii plot seeder on the 7* of June 2011. A!! herbicide treatments were applied on the 26* of July at 49 days after sowing when barley was at mid ti!!ering (approximately Z22-Z25). Herbicide treatments were applied with a spray boom delivering 100L'ha spray volume with surfactants used according to herbicide labels. Various herbicide treatments used in this study are shown in Table 1. Measurements recorded induded crop establishment, normalised difference vegetation index (NDV!), crop canopy height at maturity, grain yield, grain size and visual observations of crop damage from the treatments.

TabSe 2. Herbicide treatments investigated in BOZO barley tolerance trial.

Herijieide Rates appfjptl Siirtactart

f product fate, recommended

Coniroi (nil herbicide) 0 -

Leopard™ (quixalafop SS.Sg'L) 150, 300, 8 600 mL/ha Activator¹ ^» f0.2 % wv)

Verdict™ {haioxyfop 520% U 37.5, ZS. & 150 mUtm Uptska™ (0.5 % wv)

Fusiiade™ fftoaz/fop iSSg L) 410, 820, & MO mU e -

Correct'™ (prapsquizafap 100g L) 100, 200, & 400 f?¾Wia Hasten™

Platinum™ (cieihadsm 240g/U 125, 250, & 375 miAia Hasten™ {1 %jxv

Aramo™ (teprshxydim 200g/Q 125, 250, & 37S mU a Hasten™ (1 % -wv)

Factor™ {btiiroxydim 2S0g kg) SO, 180, & 360 ff/ha Supercharge™ (1 % wv) interv!x ^w (imazamox 3¾/L, 260, 500, & 000 mUha Superc!ia rge™ (1 % wv) Imssapyr 15 ί.}

RssuSts

Commercial cu!tivar Fleet produced about 10% higher grain yield than BOZO barley mutant in the absence of any herbicide treatment. However, application of AGCase- inhibiting FOP herbicides resulted in severe phytoioxicity to Fieet but !itiie damage to BOZO mutant barley. Application of quizalofop did not cause any detectable reduction in BOZO mutant barley yieid even at 2x rate (Figure 1 ), In contrast, even ½x caused 95% reduction in grain yield of Fleet arid a complete kill of this cu!tiyar at 1 and 2x dose of this herbicide (Figure 2). Haloxyfop showed even greater activity against Fleet with complete crop mortality at ½ x rate. BOZO mutant barley showed reduction in yield at ½x and 1x rate but there was 28% reduction at the 2x rale of ha!oxyfop. The differences in herbicide tolerance between Fleet and BOZO were also apparent for fiuazifop and propaquizafop (Figure 1).

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(Rule 91 ) ISA/AU Thsre were significant differences between Fleet and BOZO mutant barley in tolerance to three ACCase inhibiting DIM herbicides (Figure 3). Fleet showed 85-94% yield reduction even when treated with ½x dose of DIM herbicides. In contrast, BOZO mutant barley line showed no yield reduction at ½x and 1x dose of three DM herbicides (Figure 3). However, there was statistically significant yield reduction even in BOZO mutant barley at the 2x rate of these herbicides. The results clearly show that tolerance to FOP herbicides also extends to DIM herbicides.

The results from this study also showed that BOZO and Fleet are both highly sensitive to the mixture of imazamox and imasapyr (!ntervix ^m). These are imidazolines which are ALS (acetoiactate synthase enzyme) inhibitors (and are Group B or 2 herbicides). This result indicates that It would be feasible to control the volunteer plants of the BOZO line with this herbicide treatment and it would not pose any persistent weed threat to the cropping system.

Summary

Controlled environment and field studies were undertaken with BOZO barley in 2010 and 2011 to determine its response to different ACCase inhibiting herbicides. These studies have clearly shown high level of tolerance to quizalofop and other ACCase inhibiting herbicides. As expected barley eultivars Flagship and Fleet used in these studies were killed by these herbicides. Supporting studies undertaken in the field in 2011 haye shown that it is feasible to selectively control barley grass (Hordeum g!aucum) and brorne grass {Bro us rigidus) growing in BOZO barley. Studies have also shown that BOZO barley can be controlled with imidazolinone herbicides as well as giyphosate. These herbicides will prevent BOZO barley becoming a weed in following crops.

EXAMPLE 3

A range of additional experiments have been conducted to further characterise BOZO and support the results presented above. A summary of these controlled environment and field based tests follows.

2010 Program

BOZO Pot Experiment 1 (201 Q): Quizalofop at 0, 0.25, 0.5, 1.0, and 2.0 x recommended label rate (30g/ha) across SOZO mutant barley, Flagship barley cultivar and barley grass {Hordeum giaucum). Visual observations were taken.

BOZO Po _.. tperjment 2 (201_.0): The objective was to confirm tolerance to quizalofop in BOZO mutant barley and investigate tolerance to other herbicides. Herbicides included

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(Rule 91 ) ISA/AU were quizalofop, haloxyfop (ACCase-inhiblting FOP) and buiroxydim (ACCase-inhiblting DIM) at rates 0, 0,25, 0.5, 1.0 & 2.0 x recommended label rate. Both BOZO mutant barley and FLAG5HIP barley cultlvar were screened. Visual observations were made and plant biomass measured. BOZO RAG .field study (2818$: The objective was to investigate tolerance of BOZO mutant barley to quizalofop, haloxyfop and buiroxydim under field conditions at Roseworthy SA. Rates included 0, 0.5 and 1 x recommended !abe! rate and visual observations made.

2011 Program

The objective was to investigate herbicide tolerance of BOZO mutant barley to a wide range of herbicides. Herbicides included quizalofop, haloxyfop (ACCase-inhibiting FOP), c!ethodim, tepraloxydim, butrox dim (ACCase- inhiblting DIM), iodosulfuron, mesosu!furon {ALS-inhibiting sulfonylureas), imazamox /imazapyr (ALS-inhibiting imidazoiinones), and glyphosate {membrane disrupters glycines). Rates applied were 0, 0.25, 0.5, 1.0 £ 2.0 x recommended label rates and both BOZO mutant barley and FLEET barley CLiltivar were screened. Visual observations and plant biomass measurements were made.

BOZO Pot Expsrimerst 4 (2011 ): The objective was to Investigate herbicide tolerance of BOZO mutant barley to quizalofop, haloxyfop, fluazifop, propaquizafop (ACCass- inhibiting FOP), and sethoxydim (ACCase-inhibiting DIM) herbicides. Rates applied were 0, 0.25, 0.5, 10 & 2.0 x field rates and both BOZO mutant barley and Fleet barley cultlvar were screened. Visual observations and plant biomass measurements were made.

BOZQ field experiment AC-D8 (2811 ): The objective was to investigate herbicide tolerances of BOZO mutant barley and barley grass {Hordeum glaucum) control. This field site at Roseworthy SA had low fertility and treatments included quizalofop, haloxyfop, fluazifop, (ACCase-inhibiting FOP), clethodim, tepraioxydim, buiroxydim (ACCase- inhiblting DIM) herbicides at 0, 0.5, 1.0, and 2.0 x recommended label rates. Visual assessment of crop damage, barley grass control, NDV!, crap canopy height at maturity, grain yield, and grain size was measured.

BOZO field K sgrimaot AC-S2 (2811 ): reported above.

The objective was to investigate herbicide tolerance of BOZO mutant barley in a low rainfall environment and brome grass (Bromus rigidus) control. This field site had low fertility and herbicide treatments included

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(Rule 91 ) ISA/AU quizalofop, haiox fop, fluazifop, (ACCase-inhibiting FOP), cletbodirn, tepraioxydim, butroxydim (ACCase-inhibiting DIM) herbicides at 0, 0.5, 1.0, and 2.0 x field rates. Visual observations of crop damage, brome grass control, NDVi, crop canopy height at maturity, grain yield, and grain size was measured.

BOZO field experiment ..M!NTARO (2011): The objective was to Investigate herbicide tolerance of BOZO mutant barley in a high rainfall environment. Treatments investigated included quizalofop, haioxyfop, fluazifop, (ACCase-inhibiting FOP), clethodim, tepraioxydim, butroxydim (ACCase-inhibiting DIM) herbicides at 0, 0.5, 1.0, and 2.0 x field rates. Visual observations of crop damage, NDVi, crop canopy height at maturity, grain yield, and grain size was measured,

BOZQ._fiejd exp rim nt BUCKLEBOO ,12011): The objective was to investigate herbicide tolerance of BOZO mutant barley in a low rainfaii environment as well as barley grass (Hordeu glaucu n) control. Herbicide treatments were applied at 1 & 2 x recommended label rates and included quizalofop (ACCase-inhibiting FOP), c!eihoalm (ACCase-inhibiting DiM), and imazamox /imazapyr (ALS-inhibiting imidazo!inones). Crop damage was assessed based on visual symptoms and panicle density was used to determine barley grass control.

EXAMPLE 4

The inventors developed a barley variety, named 'HT017', whose phenotype is a reduced sensitivity to ACC herbicides. Seed of the variety Flagship was mutagenised with 30mM ethyl methanesuifanaie (EMS) and the ₀ and generations were grown and multiplied as unse!ected bulk populations. The M₂ generation was sown at the Chariick Experimental Farm (Strathalbyn, South Australia) on 20^t!> Ma 2009 at a density of 140 plants rrf\ The area sown was 2,5 hectares equating to approximately 40 million individual plants. The herbicide Fusilade (fluazifop) was applied a; a rate of SOOg / ha at the early tillering stage of plant development. At stem elongation surviving barley plants were transplanted to pots, grown through to maturity and grain was harvested from individual plants. Seed from the surviving plants was sown in pots in 6 ay 2010 and treated with SOQg / ha Fusilade at the 3 leaf stage of development. Assessment of survival was conducted three weeks after spraying and -99% of plants were found to be sensitive to the herbicide but the line HT017 was confirmed as tolerant to fluazifop. Figure 4 presents HT017 three weeks after treatment with fluazifop in comparison to sensitive genotypes.

Genetic analysis has identified genetic differences within the HT017 barley compared barley plants thai are ordinarily sensitive to ACC herbicides that results in an ACC

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(Rule 91 ) ISA/AU herbicide resistant phenotype. The genotype of HT017 was compared to three different barley varieties using the same methods as described above under Example 1 and a single mutation corresponding to the amino acid substitution listed in Table 3 was observed.

A single rtuc!eotide polymorphism (SNP) was identified in HT107 compared to the reference barley varieties BOZO, Alexis, Flagship and Clipper. The SMP is at base position 58277 transitioning from A to G which iesuits in an amino acid change from isoieucirie to valine (Table 3),

Tab!e 3 Summary of sequencing results showing the codons of Interest and the sequence found at that posliion in each of the five varieties, including HT107.

EXAMPLE §

The tolerance of HT017 to Fusilade (fiuaz!fep) was verified in a pot experiment. HT017 and the intolerant control barley HT066 were sown on 23^rci June 2010 with three replicates per treatment. Two treatments comprising SOOg/ha Fusilidate and 750g/ha Fusilade were applied on 23^:d July 2010 and one treatment remained unsprayed. The trial was assessed on 18^ai August 2010 and all HT066 plants were destroyed at both application rates. Ail HT017 plants survived at both application rates.

EXAMPLE 6

The mutant barley HT017 was tested for tolerance against a range of Group A herbicides. Three replicate pots were sown for each herbicide treatment of the tolerant line HT017 and also the control barley HT086. Seed was sown 17^! August 2010 and plants were

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(Rule 91 ) ISA/AU treated at the three leaf stage of plant deve!opment with the maximum !abei rate of Fusi!ade (fiuazifop), Select (ciethodim), Factor (bsjtroxydim), Verdict (ha!oxyfop). Targa (quizaiofop) or Seriin (sethoxydim). All plants of the control barley HT065 were destroyed by the treatment and the ACCase-inhibiting herbicide tolerance of HT017 was confirmed.

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(Rule 91 ) ISA/AU

Claims

CLASPS:

1 . A bariey plant, wherein said bariey plant confers resistance to inhibition by one or more acetyi-CoA carboxylase inhibiting herbicides at ieveis that would normally inhibit the growth of a barley plant.

2. A bariey plant of claim 1 , wherein the barley plan; is a commercial cu!tivar.

3. A bariey plant of claims 1 or 2, wherein the acetyi-CoA carboxylase inhibiting herbicide is selected from the group consisting of; a aryloxyphenoxypropionate herbicide and a cyclohexanediorie herbicide.

4. A bariey plant of claim 3, wherein the ary!oxyphenoxypropionate herbicide is

selected from the group consisting of; clodinafop-proparsyl; didofop-methyi, fenoxyprop-p-ethyl; quizalofop-p-ethyi; haloxyfop; fluazifop; and propaquizafop.

5. A bariey plant of claim 3, wherein the cyclohexanediorie herbicide is seiecied from the group consisting of: clethodisn; sethcxydim; iepraicxydim; tra!koxydim; and butroxydim.

6. A bariey piant of anyone of claims 1 to 5, wherein the barley plant comprises a acetyi-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: ile-_!S33Leu; Ne ^Val; and lie ;.Leu and lle _25SSVal,

7. A barley piant of anyone of claims 1 to S, wherein the barley plant comprises an acetyi-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and

» losaLeu;

* sass al;

» iajsLeu and s_t^ a!,

* SEQ iD o l ;

* SEQ ID No 2;

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(Rule 91 ) ISA/AU » SEQ ID Ho 1 and SEQ ID No 2;

SEQ ID No 3; » SEQ ID No 4; and » SEQ ID No 8.

8. A barley plant of anyone of claims 1 to 5, wherein the barley plant comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises anyone of the nucleic acids and polynucleotides selected from the group consisting of; ,₄₅₇TTA;

TTA and _e2,?GTT; SEQ ID Ho 5 SEQ ID Ho 8;

SEQ ID Nc 5 and SEQ ID No 6; and SEQ ID No 7.

9. A barley plant of anyone of claims 1 to 8, wherein the barley plant is not genetically modified.

10. A seed of a barley plant of anyone of claims 1 to 9.

1 1 . A gerrnplasm of a barley plant of anyone of claims 1 to 9.

12. A method of producing a barley hybrid with increased resisiance to inhibition by one or more acstyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant, said method comprising;

selecting a first barley plant;

selecting a second barley plant;

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(Rule 91 ) ISA/AU crossing the first barley plant with the second barley plant to produce hybrid offspring;

selecting a hybrid from said hybrid offspring with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicideacetyl-CoA carboxylase inhibiting herbicides thereby to produce the hybrid; and

wherein the second barley plant is a barley plant of anyone of claims 1 to 9.

13. A method according to claim 12, wherein the first barley plant is selected from the group consisting of AC Metcalfe, 8ass, Baudin, Buloke, CDC Copeland. CDC Reserve, Commander, Fairview, Fathom, Flagship, Fleet, Gairdner, Henley.

Hindmarsh, Keel, Navigator, Oxford, Propino, Scope, Sebastian, Skipper, SoutnemStar, Quench, V!amingh, VT Admiral, Westminster, WS4593 and Wimmera.

14. A method according to claims 12 or 13, wherein the second barley plant is selected from the group consisting of, BOZO: and HT017. 5. A barley hybrid produced from the method of anyone of ciaims 12 to 14.

18. A barley hybrid of claim 15. wherein the hybrid comprises a acetyl-CoA

carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of; lle^Leu; iie _2QS3Vai; and Ite isssL u and lie

17. A barley hybrid of claim 15, wherein the hybrid comprises a acetyi-CoA

carboxylase gene, further comprising a polynucleotide that corresponds to a carboxyl transferase domain: and

* j.ossVal:

* :₃_sLeu and a^ al;

SEQ ID No 1 ;

SEQ ID No 2;

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(Rule 91 ) ISA/AU * SEQ ID No 1 and SEQ ID No 2: SEO. ID No 3;

« SEQ ID Ho 4; and SEQ ID No 8.

18. A barley hybrid of claim 15, wherein the hybrid comprises a acetyl-CoA

carboxylase gene fiirther comprising a polynucleotide that corresponds to a carboxyl transferase domain; and

wherein the polynucleotide that corresponds to a carboxy! transferase domain comprises nucleic acids selected from the group consisting of;

• SEQ ID No 5

SEQ ID No 6,

* SEQ ID Mo 5 and SEQ ID No 8; and » SEQ ID No 7.

19. A barley hybrid according to anyone of claims 15 to 18, wherein the bariey is a commercial cu!fivar.

20. A seed of a barley hybrid according to anyone of claims 5 to 19.

21. Germp!asm of a barley hybrid according to anyone of claims 15 to 19.

22. A method of producing barley, the method comprising;

(1) selecting a barley plan! of claims 1 to 9:

(2) growing the said barley plant;

(3) harvesting barley from the barley plant; and

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(Rule 91 ) ISA AU wherein the barley plant has increased resistance to inhibition by one or more acetyl-Co.A carboxylase inhibiting herbicides ai levels that would normally inhibit the growth of a barley plant.

23. A method of producing barley, the method comprising;

(1) selecting a barley hybrid according io anyone of claims 15 to 19;

(2) growing the said barley hybrid;

(3} harvesting barley from the barley hybrid; and

wherein the hybrid has increased resistance to inhibition by one or more acety!- CoA carboxylase inhibiting herbicides at levels thai would normally inhibit the growth of a barley hybrid.

24. A method of producing barley according to claims 22. or 23, wherein the snethod improves weed control compared to weed control normally observed for producing barley.

25. A method of producing barley according to claims 22 or 23, wherein the method decreases contamination with weed species compared to contamination normally observed for producing barley.

26. A method of controlling weeds in the vicinity of a barley plant of claims 1 to 9. comprising:

(a) providing one or more acety!-GoA carboxylase inhibiting herbicides

(b) applying said one or more acetyl-CoA carboxylase inhibiting herbicides to a field comprising the barley plant of claims 1 to 9; and

(c) controlling weeds in the vicinity of said barley plant such that weed growth Is adversely affected by the application of said one or more herbicides and growth of said barley plant Is not adversely affected.

27. A method of controlling weeds in the vicinity of a barley hybrid according to anyone of claims 15 to 18, comprising:

(a) providing one or mo e acety!-CoA carboxylase inhibiting herbicides

(b) applying said one or more aeetyl~CoA carboxylase inhibiting herbicides to a field comprising the barley hybrid according to anyone of claims 15 to 19; and

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(Rule 91 ) ISA/AU (c) controlling weeds in the vicinity of said barley hybrid such that weed growth is adversely afredsd by the application of said one or snore herbicides and growth of said barley hybrid is not adversely affected.

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(Rule 91 ) ISA/AU