AU2014262183B2 - Herbicide resistant barley (2) - Google Patents

Abstract

The application discloses non-naturally occurring barley plants with increased resistance to herbicides, particularly imidazolinone herbicides. Also disclosed are the barley seeds per se, an isolated nucleic acid incorporating the nucleic acid sequence 5 conferring imidazolinone resistance, a method of inhibiting weed growth in the vicinity of a barley plant, an a method of growing a barley crop. Seeds have been deposited at NCIMB.

Description

2014262183 11 Nov 2014 P/OO/OOl Regulation 3.2 AUSTRALIA Patents Act 1990

COMPLETE SPECIFICATION STANDARD PATENT

Invention title: HERBICIDE RESISTANT BARLEY (2)

The following statement is a full description of this invention, including the best method of performing it known to us: -2-

HERBICIDE RESISTANT BARLEY (21 RELATED APPLICATIONS

This application is a divisional of Australian patent application no. 2009243917, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to grain plants having herbicide resistance or tolerance. More specifically the present invention relates to herbicide resistant barley plants.

BACKGROUND OF THE INVENTION

Food production, particularly grain production is of increasing importance worldwide due to growing populations and other pressures such as reduction in arable land, climate change and the increasing use of crops for biofuel production.

Herbicides play an important role in modern agriculture by maximizing grain production, pasture production, quality and profitability. Herbicides allow undesirable plants to be killed or suppressed and thereby reduce competition with desirable plants such as crop plants for nutrients, water and the like.

Imidazolinone and sulphonylurea herbicides inhibit the growth and development of susceptible plants, including a wide range of weeds, by inhibiting the plant enzyme Acetohydroxy acid synthase (AHAS), also known as acetolactate synthase (ALS). AHAS synthesizes branch chain amino acids, a key function in plants. Importantly, imidazolinone and sulphonylurea herbicides also have relatively low toxicity to animals, including humans.

Imidazolinones include the following compounds imazethapyr, imazaquin, imazapyr, imazapic, imazamox and the like.

Commercially available imidazolinone herbicides include ON DUTY® (imazapic + imazapyr) INTERVIX® (imazamox + imazapyr) SPINNAKER® (imazethapyr), RAPTOR® (imazamox) and FLAME® (imazapic).

Although naturally occurring resistance to herbicides has been described in some crops many important food crops are susceptible to herbicides including imidazolinone.

Barley (Hordeum vulaare) is an important grain crop internationally because it is used in malt production, brewing and for human and animal feed. It has the advantage of being more salt tolerant than wheat and can be grown successfully on poorer quality land and/or in drier environments.

Weed control is a major issue in barley production. Good chemical weed control is desirable to maximize production of the crop and limit the need for mechanical cultivation -3-and resultant damage to soil structure and erosion. In particular it would be desirable to utilize a greater range of low toxicity herbicides to control the full spectrum of a variety of weeds in a barley cropping system. For example, as most barley is resistant to sulphonylureas, this herbicide can be applied post emergence to growing barley crops, however, imidazolinones cannot be applied post emergence because barley is susceptible to this class of herbicides. Further, the crop cannot even be sown into soil containing imidazolinones because of its susceptibility to this herbicide.

The above references to and descriptions of prior proposals or products are not intended to be, and are not to be construed as, statements or admissions of common general knowledge in the art.

SUMMARY OF THE INVENTION

The invention provides a non-naturally occurring barley plant which has increased resistance to herbicides compared with the wild type barley.

In a preferred embodiment, the present invention provides a non-naturally occurring barley plant with increased resistance to imidazolinone herbicides compared to a wild type barley plant and having the herbicide resistance characteristics of NCI MB Deposit accession number NCIMB 41547 or NCIMB 41551 or a derivative of said NCIMB Deposit accession number having the imidazolinone resistance characteristics thereof.

In another preferred embodiment, the present invention provides a non-naturally occurring barley plant with increased resistance to imidazolinone herbicides compared to a wild type barley plant and as deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551.

In a further preferred embodiment, the present invention provides a non-naturally occurring barley plant with increased resistance to imidazolinone herbicides compared to a wild type Buloke barley plant and deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551.

The invention also provides a seed of the non-naturally occurring barley plant.

Preferably, the seed has the imidazolinone resistance characteristic thereof.

In a preferred embodiment, the present invention provides the seed or a derivative of NCIMB deposit accession number NCIMB 41547 or NCIMB 41551 having the imidazolinone resistance characteristics thereof.

Further the invention provides a method of inhibiting weed growth in the vicinity of a barley plant comprising growing a barley plant with increased resistance to a herbicide compared to a wild type barley plant and applying under suitable conditions imidazolinone to said barley plant with increased resistance and its vicinity sufficient to inhibit weed growth. -4- ln a preferred embodiment, the present invention provides a method of inhibiting weed growth in the vicinity of a barley plant comprising growing a barley plant with increased resistance to a herbicide compared to a wild type barley plant and applying under suitable conditions imidazolinone to said barley plant with increased resistance and its vicinity sufficient to inhibit weed growth, wherein said barley plant has the imidazolinone resistance characteristics of NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551 or is a derivative of said NCIMB Deposit accession number having the imidazolinone resistance characteristics thereof.

In another preferred embodiment, the present invention provides a method of inhibiting weed growth in the vicinity of a barley plant comprising growing a barley plant with increased resistance to a herbicide compared to a wild type barley plant and applying under suitable conditions imidazolinone to said barley plant with increased resistance and its vicinity sufficient to inhibit weed growth, wherein said barley plant is as deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551 or is a derivative of said NCIMB Deposit accession number having the imidazolinone resistance characteristics thereof.

The invention also relates to a method of growing a barley crop comprising sowing the seeds of the invention and cultivating the seeds and resultant plants under suitable conditions to produce a crop.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a sequence alignment for the AHAS gene localised around base 1742 (indicated by a box) of the following imidazolinone resistant mutant lines: VBHT 0805 (SEQ ID NO: 6) VBHT 0806 (SEQ ID NO: 7) VBHT 0802 (SEQ ID NO: 8) and the following wild type comparators: AF059600 (SEQ ID NO: 3)

Buloke (SEQ ID NO: 4)

Hindmarsh (SEQ ID NO: 5).

Figure 2 shows amino acid sequence alignment of the AHAS gene for six plant species localised around mutant DNA nucleotide base 1742 identified in lines EMS05*06HI005 and EMS05*06HI006 (H.vulgare_AHAS_mutant) and the resulting alteration of a Serine (S) to an Asparagine (N):

Nicotiana tabacum AHAS/1-24 (SEQ ID NO: 9)

Brassica napus AHAS1/1-24 (SEQ ID NO: 10) -5-

Brassica napus AHAS2/1-24 (SEQ ID NO: 11)

Hordeum vulgare AHAS/1-24 (SEQ ID NO: 12)

Hordeum vulgare AHAS mutant/1-24 (SEQ ID NO: 13)

Oryza sativa AHAS/1-24 (SEQ ID NO: 14)

Triticum aestivum AHAS/1-24 (SEQ ID NO: 15)

Zea mays AHAS/1-24 (SEQ ID NO: 16).

Figure 3 shows alignment of resequenced AHAS gene for VBHT 0805, VBHT 0806, VBHT 0802 and VBHT 0810 compared to the wild type reference sequences (AHAS_H.vulgare_AF059600 and Buloke): VBHT 0805 (SEQ ID NO: 1) VBHT 0806 (SEQ ID NO: 2) VBHT 0802 (SEQ ID NO: 17) AHAS H. vulgare AF059600 (SEQ ID NO: 18) VBHT 0810 (SEQ ID NO: 19)

Buloke (SEQ ID NO: 20)

Figure 4 shows the imidazolinone resistance of the mutated lines VBHT0802, VBHT0805, VBHT0806 and VBHT0810 compared to the wild type lines Buloke and Hindmarsh in experiments at Horsham, Victoria, Australia in 2007 (Table 3), and 2008 (Table 4) and at Marinna, NSW, Australia in 2008 (Table 5). The experiments are described in Example 5.

Figure 5 shows in Table 6 yield (t/ha) of imidazolinone resistant mutant lines compared with Buloke at 9 sites in Australia. The experiments are described in Example 6.

Figure 6 shows in Table 7 morphological and DNA comparison of imidazolinone resistant mutant barley lines with parent cultivar Buloke. The experiments are described in Example 7.

Figure 7 shows in Table 8 malting quality for VBHT0805 compared to Buloke. The experiments are described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a non-naturally occurring barley plant which has increased resistance to herbicides compared to wild type barley, particularly resistance to imidazolinone herbicides. Progeny of the barley plants and descendants having the -6-increased herbicide resistance trait or characteristic are also included in the invention. The invention also includes hybrids of the barley plants of the invention.

The term “non-naturally occurring” refers to a plant which does not occur in nature and is the result of human intervention. Preferably the plants are produced by mutagenesis, more preferably chemically induced mutagenesis.

The term “barley plant” refers to the species Hordeum vulgare in its total genetic variation and its many varieties such as Buloke and Hindmarsh, and to related species within the Hordeum genus.

The terms “increased resistance to herbicides” and “herbicide resistant barley” mean that the plant is not as adversely affected by the herbicide as its naturally occurring counterpart or wild type. The term includes tolerance to herbicides.

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

While not wishing to be bound by theory, it appears that the increased herbicide resistance developed by the present inventors’ results from changes to the physiology and/or biochemistry of the plant and thus interferes with the normal action of the herbicide. This may involve altered absorption and/or translocation of the 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 protein(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 plants and seeds of the invention include plants and seeds that are mutations, recombinant variants and genetically engineered derivatives or derivatives derived by other means such as conventional breeding, which retain the altered elements described above. The invention also extends to plant parts other than seeds of the herbicide resistant barley, including plant cells, plant tissues and plant organs whether produced by plant tissue culture or otherwise.

In a preferred embodiment the seeds of the invention are VBHT 0802, VBHT 0803, VBHT 0805, VBHT 0806, VBHT 0807, VBHT 0809 or VBHT0810 as described herein or are plants derived from these seeds. The invention also provides seeds or plants which are mutants, derivatives and variants thereof having increased imidazolinone resistance compared to wild type barley plants.

Samples of the following seeds were deposited in accordance with the Budapest Treaty on 18 April 2008 at NCIMB Ltd of Ferguson Building, Craibstone Estate, Buckburn, Aberdeen, Scotland and accorded accession numbers as shown: -7-

Table 1: Deposits

Designation Accession No. VBHT 0802 NCIMB 41547 VBHT 0803 NCIMB 41548 VBHT 0805 NCIMB 41549 VBHT 0806 NCIMB 41550 VBHT 0809 NCIMB 41551

The invention also provides a method of inhibiting weed growth in the vicinity of a barley plant comprising growing a barley plant with increased resistance to a herbicide compared to a wild type barley plant and applying under suitable conditions imidazolinone to said barley plant and vicinity sufficient to inhibit weed growth.

The invention also relates to a method of inhibiting weed growth in a barley crop said method comprising growing a herbicide resistant barley crop and applying a imidazolinone herbicide to said crop under suitable conditions to inhibit weed growth.

The imidazolinone may be used as a combination of imazamox and imazapyr at a rate of 12.375 g per hectare and 5.625 g per hectare respectively; 24.75 and 11.25 g per hectare respectively; 41.25 and 18.75 g per hectare respectively or 49.5 and 22.5 g per hectare respectively. Alternatively, the imidazolinone may be used as a combination of imazapic and imazapyr at a rate of 26.25 and 8.75 g per hectare respectively or 42 and 14 g per hectare respectively.

The invention also relates to a method of growing a barley crop comprising sowing the seed of the invention and cultivating the seed and resultant plant under suitable conditions to produce a crop.

Preferably the herbicide resistant barley used for the crop is produced by mutagenesis, more preferably chemical mutagenesis.

Still more preferably the herbicide resistant crop is, or is derived from, NCIMB 41547, NCIMB 41548, NCIMB 41549, NCIMB 41550 or NCIMB 41551 or has the herbicide resistant characteristics thereof.

In this specification, the term ‘comprises’ and its variants are not intended to exclude the presence of other integers, components or steps.

The following non limiting examples describe the invention.

Example 1: Production of Mutants 33.3kg (approximately 812,195 seeds) of the barley variety Buloke was soaked in 0.25% Ethyl methane sulfonate (EMS) and dried using an air blower. The parent Buloke is a high yielding variety which produces grain of malting quality. Surprisingly Buloke is -8- suited to areas where brome grass is a problem weed. In selecting this strain, the inventors hoped to develop an imidazolinone resistant barley variety which could be grown under conditions where brome grass and other weeds may be controlled with imidazolinone herbicides.

Example 2: M1 generation

Mutated barley seed from Example 1 was sown in a plot of 0.5ha at Horsham, Victoria, Australia in 2006. Management was consistent with those recommended for barley in the region. At maturity the plot was harvested by an experimental plot harvester. 300g samples were taken from each full seed bin and bulked together to form a representative smaller composite (150kg) and remaining seed was bulked together as a composite sample (600kg).

Example 3: M2 generation 200kg of mutated barley seed from example 2 (approx. 4.88 million seeds) was sown on 2 ha of land at Horsham, Victoria, Australia in 2007. Emerged plants were sprayed with ON DUTY® at a rate of 80g/ha (active ingredient. Imazapic 42g + Imazapyr 14g) and 20 surviving plants were harvested individually by hand and retained.

Example 4: Preliminary evaluation of M2 selections for resistance to Imidazolinone herbicides.

The ten plants with the largest dry matter and seed yield from Example 3 were identified and designated BULOKE-EMS05*06HI001, BULOKE-EMS05*06HI002, BULOKE-EMS05*06HI003, BULOKE-EMS05*06HI004, BULOKE-EMS05*06HI005, BULOKE-EMS05*06HI006, BULOKE-EMS05*06HI007, BULOKE-EMS05*06HI008, BULOKE-EMS05*06HI009, BULOKE-EMS05*06HI010. 30 seeds from each selection were sown in 10 pots (3 seeds per pot) in a glasshouse. Half of the pots for each selection were sprayed with ON DUTY® at a rate of 50g/ha (active ingredient. Imazapic 26.25g + Imazapyr 8.75g) and each selection classified for resistance to ON DUTY® based on the reaction of progenies. Plants were deemed resistant if they appeared unaffected by ON DUTY® (no visual symptoms) or susceptible if they were killed by ON DUTY®, • The following Homozygous susceptible (escapes in field screening) selections were identified and discontinued from further evaluation: BULOKE-EMS05*06HI001, BULOKE-EMS05*06HI004, BULOKE-EMS05*06HI008. 2014262183 05 Jan 2017 - 8a - • The following Homozygous resistant selections were identified and resistant plants bulked together for evaluation: BULOKE-EMS05*06HI002, BULOKE-EMS05*06HI005, BULOKE-EMS05*06HI006, BULOKE-EMS05*06HI009, BULOKE- 5 EMS05*06HI010. • Although homozygous for resistance to ON DUTY, BULOKE-EMS05*06HI009 was segregating for genes that affected plant type and single plants were harvested separately for further evaluation. • Heterozygous susceptible/resistant selections were identified and individual 10 resistant plants were harvested for further evaluation: BULOKE-EMS05*06HI003, BULOKE-EMS05*06HI007 -Θ Α homozygous herbicide resistant line representing each original resistant M2 plant selection was selected for submission to NCIMB and further evaluation. These lines were given shorter, new names to improve communications as shown in Table 2:

Table 2: Original and new names for homozygous herbicide resistant lines

Name Original name VBHT0802 BULOKE-EMS05*06HI002 VBHT0803 BULOKE-EMS05*06HI003-06GI003 VBHT0805 BULOKE-EMS05*06HI005 VBHT0806 BULOKE-EMS05*06HI006 VBHT0807 BULOKE-EMS05*06HI007-06GI001 VBHT0809 BULOKE-EMS05*06HI009-06G082 VBHT0810 BULOKE-EMS05*06HI010

Example 5: Field evaluation for tolerance to Intervix a) Selection of lines with homozygous resistance to Imidazolinone herbicides

Seed from single plants selected in the glasshouse (Example 4) were sown in 5m paired rows at Horsham, Victoria, Australia, in 2007 and sprayed with 0.75L/ha of Intervix® (active ingredient Imazamox 33grams/kg + Imazapyr 15 grams/kg) post emergence and rows that were visually unaffected by the herbicide were harvested for further evaluation (data not presented). Homozygous imidazolinone/esistant lines VBHT0803 and VBHT0806 were identified and submitted to NCIMB. b) Evaluation of resistance to Imidazolinone herbicides

The imidazolinone resistance of the mutated lines VBHT0805, VBHT0806, and VBHT0810 were compared to the wild type lines Buloke and Hindmarsh in experiments at Horsham, Victoria, Australia in 2007 and 2008 and Marinna, NSW, Australia in 2008. At Horsham the experiments were sown in a split plot design with 2 replications in 2007 and 3 replications in 2008, and at Marinna a factorial design was used with 3 replications. At Horsham genotypes (Selections) were allocated as subplots and herbicide treatment as mainplots Herbicide treatments were: o Horsham 2007- no applied Intervix® and Intervix® applied post emergence at 0.75L/ha (active ingredient24.75g imazamox + 11.25g imazapyr) o Horsham 2008 - no applied Intervix® and Intervix® applied post emergence at 0.375L/ha (active ingredient 12.375g imazamox + 5.625g imazapyr), - 10- 0.75L/ha (active ingredient 24.75g imazamox+ 11.25g imazapyr), 1.5L/ha (active ingredient 49.5g imazamox + 22.5g imazapyr). o Marinna 2008 - no applied Intervix® and Intervix® applied post emergence at 0.375L/ha (active ingredient 12.375g imazamox + 5.625g imazapyr), 0.75L/ha (active ingredient 24.75g imazamox + 11.25g imazapyr), 1.25L/ha (active ingredient 41.25g imazamox + 18.75g imazapyr).

Management of all experiments was consistent with established commercial practices except the application of Intervix® herbicide. Plots were harvested using a small plot harvester, and seed weighed, converted to yield in t/ha and analyzed using REML. A 500 gram sample from replication 1, no applied Intervix® was evaluated for malting quality using established methods (see Example 10).

Intervix® applied at all rates significantly reduced the grain yield of the varieties Buloke and Hindmarsh compared to the control treatment (no applied Intervix) in all three experiments (Tables 3, 4 and 5). In both cases all plants in the Intervix® treated plots turned yellow, ceased to grow and died. In contrast, there was no significant difference in the grain yield of VBHT0805, VBHT0806 or VBHT0810 when treated with Intervix® at any rate compared to the control treatment (no applied Intervix). Furthermore, no visual symptoms of damage could be observed in the Intervix® treated plots of VBHT0805, VBHT0806 orVBHT0810. VBHT0805, VBHT0806 or VBHT0810 are all derived from Buloke that was treated with EMS and clearly exhibit improved resistance to Intervix® at rates from 0.375L/ha (active ingredient 12.375g imazamox + 5.625g imazapyr) to 1.5L/ha (active ingredient 49.5g imazamox + 22.5g imazapyr) compared to the wild type parent Buloke and the cultivar Hindmarsh.

Example 6: Field evaluation to compare the yield of imidazolinone resistant mutant barley lines with Buloke.

The yield (t/ha) of imidazolinone resistant mutant lines VBHT0805, VBHT0806 or VBHT0810 were compared with Buloke at 9 sites in Australia.

Paskeville and Callington were located in the state of South Australia, Forbes, Temora and Junee Reefs in New South Wales, Dimboola, Elmore and Mt Mercer in Victoria, and Northam early and late sown in Western Australia. VBHT0805 and VBHT0802 had the highest yields across all sites and averaged 4% higher than Buloke indicating that the mutation process had not cause deleterious mutations that - 11 - significantly affect grain yield in Australia. Alternatively VBHT0806 was 4% lower yielding than Buloke. These results are shown in Table 6.

Example 7: Morphological and DNA comparison of imidazolinone resistant mutant barley lines with Buloke

The morphological characteristics and DNA of imidazolinone resistant mutant barley lines VBHT0802, VBHT0803, VBHT0805, VBHT0806, VBHT0807, VBHT0809 and VBHT0810 were compared with the wild type Buloke. Table 7 describes the distinctive morphological and DNA characteristics identified. VBHT0803 differs from Buloke in having purple awns and is shorter than Buloke but not a dwarf. VBHT0809 has purple awns and is a dwarf plant with a prostrate vegetative growth habit. VBHT0805, VBHT0807 and VBHT0810 have identical haplotypes suggestive of being derivatives of a single mutational event. VBHT0802, VBHT0803, VBHT0806 and VBHT0809 all have unique haplotypes that is likely to be due to the heterogeneity within the original Buloke sample used for the mutagenesis and indicates that each represents a unique mutational event for resistance to imidazolinone herbicides. The haplotypes of VBHT0805, VBHT0807 and VBHT0810 differed from the Buloke reference sample (VB0105*12) at 41 of 1424 single polynucleotide polymorphism (SNP) loci.

Example 8: DNA sequencing of the AHAS gene of imidazolinone resistant mutant barley lines. a. Overview:

Mutant lines of barley (Hordeum vulgare L.) screened for resistance to the herbicide imidazolinone and identified as containing resistance determinants as per the previous examples were tested to confirm and characterise the mutant lines, the suspected mutant alleles of the acetohydroxyacid synthase (AHAS) gene, and resequenced to identify DNA base changes. The DNA sequences were collated and compared to wild type sequences and known mutations. b. Experimental Approaches and Results:

The plant material consisted of the wild type genotypes, Buloke along with 4 mutant lines derived from Buloke numbered VBHT0802 (NCIMB 41547), VBHT0805 (NCIMB 41549), VBHT0806 (NCIMB 41550) and VBHT0810 (not deposited). A collection of publicly available reference sequences of the AHAS gene was assembled from Genbank. The sequences were obtained from a range of monocotyledonous species, (including barley) and were used for PCR primer design to conserved regions of the gene to ensure optimal primer performance. A total of 20 primers were designed in forward and reverse orientation - 12- to enable maximal coverage of the available gene sequence and provide multiple options for primer pairing to mitigate the possibility of poor primer performance. PCR amplification using standard conditions was tested on all supplied DNA templates. Amplification performance was low, with all possible primer combinations and resulted in the production of multiple products as resolved on an agarose gel. Nested PCR was employed to reduce amplification product complexity and to increase reaction specificity. Using the nested PCR strategy a region of the AHAS gene was uniquely amplified and was subjected to direct sequencing using BigDye 3.1 resolved on an Abi 3730x1. The sequences were then aligned using Sequencher v3.7 and assessed for polymorphism.

Several variant mutated bases were identified. At base coordinate 1431 line VBHT0810 has a transition from G to A. However, this change is unlikely to be causal for the observed phenotype of herbicide resistance as this specific nucleotide is the third base in the triplet codon and codes for the same amino acid and is therefore synonymous.

In lines VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB 41550) at base coordinate 1742 G has been mutated to A, also a transition. This causes alteration of the resulting amino acid from serine to asparagine (Figure 1).

Using cross-species comparative analysis, the serine amino acid that has been mutated in lines VBHT0805 (NCIMB 4159) and VBHT0806 (NCIMB 41550) has been identified as being highly conserved in other species (Figure 2).

Comparison of this serine to asparagine specific change to known causal mutations that confer plant herbicide resistance, conforms to previously identified and characterised changes (Li et al 2008; Jander et al. 2004; Sathasivan et al. 1990). c. Conclusions:

Mutant lines VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB 41550) are likely to express herbicide resistance due to alteration of coordinate 1742 changing from a G to an A, which alters the amino acid from a serine to an asparagine. This mutation is referred to as SER653. The imidazolinone herbicide resistance shown in the barley of the present invention is surprisingly high compared to levels found in other species. This was unexpected and while not wishing to be bound by theory it may be possibly due to the high copy number of the AHAS gene in barley.

The mutation(s) causing herbicide resistance in lines VBHT0802, VBHT0803, VBHT0807, VBHT0809 and VBHT0810 are currently unknown. Mutations are likely to be in the unsequenced part of the AHAS gene and may have alternate mechanisms for resistance. VBHT0802 has a different genetic control for resistance than VBHT0805 and VBHT0806 and does not have the mutation SER653. 2014262183 11 Nov 2014 - 13-

Example 9: Further sequencing of VBHT0802, VBHT0803, VBHT0805, VBHT0806, VBHT0807, VBHT0809 and VBHT0810

Further work is being carried out using standard sequencing techniques similar to those of Example 8 to characterise the AHAS gene and other possibly relevant genes 5 involved in conferring imidazolinone resistance in VBHT0802 (NCIMB 41547), VBHT0803 (NCIMB 41548), VBHT0805 (NCIMB 41549), VBHT0806 (NCIMB 41550), VBHT0807 (not deposited), VBHT0809 (NCIMB 41551) and VBHT0810 (not deposited).

Even though the SER653 mutation has been identified in VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB 41550), these are included in the further sequencing since to it is believed there may be another mutation in the part of the gene that was not sequenced.

Example 10: Suitability for malt production.

Buloke is currently recognised as a malting cultivar in Australia. Tests for suitability for malt production were carried out in accordance with the standard procedures of the 15 European Brewing Congress. The results shown in Table 8 demonstrate that VBHT0805 (NCIMB 41549) is equivalent to Buloke in its suitability for malting.

Example 11: Production of herbicide resistant derivates of VBHT0805 via backcrossing with Hindmarsh. 20 The imidazolinone resistance from VBHT0805 has been transferred through backcrossing to derivatives of the high yielding dwarf barley variety Hindmarsh that is susceptible to imidazolinone herbicides. Lines from these crosses have been identified that retain the characteristics of Hindmarsh, including dwarf growth habit, but also have resistance to imidazolinone herbicides that is comparable to VBHT0805. Hindmarsh is 25 genetically different and unrelated to Buloke, the original line used to develop VBHT0805, and these findings indicate that the imidazolinone resistance discovered and outlined in the above examples can be transferred to progeny and are thus derivatives of VBHT0805. 2014262183 11 Nov 2014 - 14-

References

Li D., Barclaya I., Jose K., Stefanova K., Appels R. 2008. A mutation at the Ala122 position of acetohydroxyacid synthase (AHAS) located on chromosome 6D of wheat; improved 5 resistance to imidazolinone and a faster assay for marker selection. Mol Breeding. Published Online early.

Jander G., Baerson S., Hudak J., Gonzalez K., Gruys K., Last R. 2003 Ethylmethanesulfonate Saturation Mutagenesis in Arabidopsis to Determine Frequency of to Herbicide Resistance. Plant Physiology131 p139-16.

Sathasivan K., Haughn G., Murai N. 1990 Nucleotide sequence of a mutant acetolactate synthase gone from an imidazolinone-resistant Arabidopsis thaliana var. Columbia. Nucleic Acids Research 18 2188. 15

Claims (18)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A non-naturally occurring barley plant with increased resistance to imidazolinone herbicides compared to a wild type barley plant and as deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551.
2. 2. A seed of the plant of claim 1 and having the imidazolinone resistance characteristics thereof.
3. 3. A barley plant, or a descendent of a barley plant, grown or otherwise derived from the seed of claim 2 and having the imidazolinone resistance characteristics thereof.
4. 4. The seed or a derivative of NCIMB deposit accession number NCIMB 41547 or NCIMB 41551 having the imidazolinone resistance characteristics thereof.
5. 5. A barley plant derived from the seed or derivative of claim 4, or a plant descendant of the seed or derivative, and having the imidazolinone resistance characteristics thereof.
6. 6. A non-naturally occurring barley plant with increased resistance to imidazolinone herbicides compared to a wild type Buloke barley plant and as deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551.
7. 7. A non-naturally occurring barley plant according to claim 6, wherein said barley plant is as deposited under NCIMB Deposit accession number NCIMB 41547.
8. 8. A non-naturally occurring barley plant according to claim 6, wherein said barley plant is as deposited under NCIMB Deposit accession number NCIMB 41551.
9. 9. A seed of the plant of any one of claims 6 to 8 and having the imidazolinone resistance characteristics thereof.
10. 10. A barley plant grown from the seed of claim 9 and having the imidazolinone resistance characteristics thereof.
11. 11. A method of inhibiting weed growth in the vicinity of a barley plant comprising growing a barley plant with increased resistance to a herbicide compared to a wild type barley plant and applying under suitable conditions imidazolinone to said barley plant with increased resistance and its vicinity sufficient to inhibit weed growth, wherein said barley plant is as deposited under NCIMB Deposit accession number NCIMB 41547 or NCIMB 41551 or is a derivative of said NCIMB Deposit accession number having the imidazolinone resistance characteristics thereof.
12. 12. A method of inhibiting weed growth in the vicinity of a barley plant comprising growing the barley plant of claim 1, 5, 6, 7 or 8, or growing a barley plant from the seed of claim 2, 4 or 9, and applying under suitable conditions imidazolinone to said barley plant and its vicinity sufficient to inhibit weed growth.
13. 13. The method of claim 11 or claim 12, wherein imidazolinone is used as a combination of imazamox and imazapyr applied post emergence.
14. 14. The method of claim 13, wherein the imazamox and imazapyr are applied at a rate of 12.375 g per hectare and 5.625 g per hectare respectively; 24.75 and 11.25 g per hectare respectively; 41.25 and 18.75 g per hectare respectively or 49.5 and 22.5 g per hectare respectively.
15. 15. The method of claim 11 or claim 12, wherein imidazolinone is used as a combination of imazapic and imazapyr.
16. 16. The method of claim 15, wherein the imazapic and imazapyr are applied at a rate of 26.25 and 8.75 g per hectare respectively or 42 and 14 g per hectare respectively.
17. 17. A method of growing a barley crop comprising sowing the seed of claim 2, 4 or 9, and cultivating the seed and resultant plant under suitable conditions to produce a crop.
18. 18. The method of claim 17, wherein an imidazolinone herbicide is applied at suitable rates and at suitable times to inhibit growth of weeds in the crop.