Imidazolinone herbicide resistant sugar beet plants

background the invention

(1) Summary of invention

The The present invention relates to a method for producing sugar beet plants (Beta vulgaris L.) to imidazolinone herbicides for weed control be employed are resistant. In particular, referred to in the present invention relates to sugar beet plants, consisting of a derived susceptible sugarbeet were by selection of a mutation of a acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), coding Gene using the herbicide with cells in a culture medium.

(2) Description of the Prior of the technique

Of the Prior art has the genetic modification of the acetolactate synthase gene with By recombinant means, such as in US Patents No. 5,013,659.; 5,141,870; and 5,378,824 at Bedbrook et al. shown will be described. This type of modification in sugar beet plants, in the example IV in Patent '824 shown. The results were less than satisfactory in the production of plants, the purebred herbicide resistance passed on.

A Variety resistant plants were produced and crossed. Saunders et al. also describe in Crop Science 32: 1357-1360 (1992) the production of the sugar beet plant (CR1-B) which is opposite Sulfonylureas is resistant, autogamous from receptive Clone (REL-1) by selection of mutation cells in a culture medium, containing the herbicide. Various resistant plants were produced and crossed. Hard et al. (Weed Sci. 40: 378-383 (1992); and Weed Sci. 41: 317-324 (1993)) further characterized the resistant line and determined, that resistance to changes ALS activity was due and no cross-resistance to other ALS-inhibiting herbicides showed and was encoded by a single, semidominant gene.

It There are no publications, describe an imidazolinone resistance obtained by modifying ALS or AHAS in sugar beet plants has been obtained. Different grain lines with this resistance have been developed, such as Newhouse et al. in Theoretical and Applied Genetics 83: 65-70 (1991) describe.

On commercial way to securing the harvest is the use of "safe ners" as in the European Patent No. 375.875 are described. This method requires a more Chemical into the soil. The preferred method is, sugar beet plants to develop that are resistant to imidazolinone herbicides.

tasks

It is therefore an object of the present invention, a method to disposal ask to for imparting resistance to imidazolinone herbicides on sugar beet plants by selection of a mutation of a acetolactate synthase gene encoding this resistance coded. It is further an object of the present invention, Sugar beet plants to disposal questions to which are resistant to this herbicide. These and Other objects will be apparent to an increasing extent with reference to the following description and figures.

Short description the pictures

<figref idrefs="S17">1</figref> provides a schematic view of the method of the present invention for producing imidazolinone resistant sugar beet plants is R = resistant. IM = imidazolinone; S = sensitive; SU = sulfonylurea; Sur = ALS Allele dominant for SU-R, TP-R and IM-S; SIR-13 = ALS allele dominant for SU-S, TP-S, and IM-R; TP = triazolopyrimidine. REL-1 was the starting material for obtaining the imidazolinone resistance.

<figref idrefs="S18">2</figref> is a flow diagram for generating a the detailed steps mutant sugar beet plant shows.

<figref idrefs="S19">3</figref> is a graph against the imazethapyr resistance spray application postemergence &lt;?page 3?&gt;on sugar beet plants shows.

<figref idrefs="S20">4</figref> is a graph illustrating the imazethapyr resistance in an ALS assay shows in a cell culture.

description of the Preferred Embodiments

The This invention relates to a sugar beet plant material, consisting of mutated cells with a mutated acetolactate synthase gene is, encoding the synthase, wherein the mutated cells have a resistance to an imidazolinone herbicide, and said Resistance by conventional cross-breeding of plants produced from these Cells have been generated, transferable is.

furthermore The present invention relates to a method for producing a herbicide resistance in a sugar beet plant which comprises: In Exposing first cells of the sugar beet to an imidazolinone herbicide, for the the first cells are sensitive in a culture medium; and selecting second cells having the herbicide resistance, wherein the Resistance by cross-breeding of plants containing the second cells included, transmitted can be.

Finally relates the present invention is directed to a method for transmitting a herbicide resistance in a sugar beet which comprises: contacting Exposing first cells of sugar beet plant to an imidazolinone herbicide, for the the first cells are sensitive in a culture medium; Select second Cells having the herbicide resistance; planting a first plant from the second cells so that the plant which having herbicide resistance, and; Crossing the first plant with second plants to produce crossed plants which have the having herbicide resistance.

The Sugar beet plant REL-1 (Regenerating, East Lansing-1) is available from Dr. Joseph Saunders, Research Geneticist, US Department of Agriculture, East Lansing, Michigan and 1987 public accessible. It is available for free. REL-1 is sensitive to Imidazolinone (IM-S) sulfonylurea (SU-S) and Triazolopyridinsulfonamid- (TP-S) Herbicides, such as in <figref idrefs="S17">1</figref> is shown.

Among Using callus and suspension cultures, a line developed that is heterozygous for imidazolinone resistance (IM-R) was. This cell line (seed) was under the Budapest Agreement as ATCC 97534 with the American Type Culture Collection filed on May 6, 1996 and is referred to herein as Sir-13th The cell line is indicating the name and the number on request available, However, no license has been issued on the basis of such deposit. By breeding with elite sugar beet plant lines, particular Beta vulgaris L., especially commercially can useful, resistant sugar beet plants be generated. Traditional breeding techniques can be used to the imidazolinone resistance to other beets such as eg beetroot to transfer.

Sugar beet plants, in violation of the imidazolinone herbicide, especially against imazethapyr, are resistant, solve the problem of soil residues due the use of this herbicide in sugar beet growing areas. Presently there there is a waiting period of 40 months between the use of this Herbicide and the future Use of that field for growing sugar beet plants. Secondly allowed a high level of imidazolinone herbicide resistance to the use of an imidazolinone to combat of weeds in sugar beet.

the Example 1 below shows the isolation and breeding of a novel sugar beet plant (Sir-13).

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EXAMPLE 1

1. Selecting Imidazolinone resistant sugar beet variants (Sir-13).

Description of the material

<ul><li><dl><dt>Rel-1</dt><dd>(Regenerating East Landing-1) is a hochregenerierbare male-fertile sugar beet line, the for the initial Cell selection for Imidazolinone herbicide-resistant Mutant is used, and is used as a sensitive control for most Experiments used. </dd><dt>Sir-13</dt><dd>This Zuckerrübenisolat was containing by plating Rel-1 cells on imazethapyr a Medium developed. This variant is imidazolinone resistant but not cross-resistant to sulfonylureas</dd><dt>Sir-30</dt><dd>A representative selected Isolate which tends to be infertile. This was an aberrant Result of the cell culture process.</dd></dl></li></ul>

<ul><li>a. Plants were selected as starting materials for a somaclonal selection based on their ability, under Kalli High cytokinin conditions to generate (in B1 media) and scions or engines of the Kalli regenerate.</li></ul>

Protocol for B1 media

<ul><li>30 g / l sucrose</li><li>100 mg / l myo-inositol</li><li>1.65 g / l NH₄NO₃</li><li>1.90 g / l KNO₃</li><li>0.44 g / l CaCl₂· 2H₂O</li><li>0.37 g / l MgSO₄· 7H₂O</li><li>0.17 g / l KH₂PO₄</li><li>6.2 mg / l H₃BO₃</li><li>16.8 mg / l MnSO₄·H₂O</li><li>10.6 mg / l ZnSO₄· 7H₂O</li><li>0.88 mg / l Kl</li><li>0.25 mg / l Na₂MoO₄· 2H₂O</li><li>0.025 mg / l CuSO₄· 5H₂O</li><li>0.025 mg / l CoCl₂· 6H₂O</li><li>37.3 mg / l Na₂EDTA</li><li>27.8 mg / l FeSO₄· 7H₂O</li><li>1 mg / l thiamine</li><li>0.5 mg / l pyridoxine</li><li>0.5 mg / l nicotinic acid</li></ul>

1 mg / l benzylaminopurine

<ul><li>pH 5.95</li></ul>

Celebrations B1 media were autoclaved with 9 g / l and with Pflanzenkulturagar a filter Sterilized herbicide stock solutions as for the selection scheme needed modifziert. The agar medium was in 15 × 100 mm disposable plastic petri dishes cast. Liquid B1 media was in aliquots of 40 ml in 125 ml Erlenmeyer flask and split for application to liquid Suspension cultures autoclaved.

to Selection of the imidazolinone resistant variant, Sir-13, which was Feedstock Rel-1. <ul><li>b. To select the Imidazolinone resistant variant of a procedure was followed, in the <figref idrefs="S18">2</figref> will be shown.</li></ul>

Leaf disk explants from rapidly expanding leaves the original plant were prepared. The foliage were surface sterilized (Step 1) acetic by two successive 20-minute washes with 15% commercial bleach plus 0.025% Triton X-100 (T-9284, Sigma Chemical Co., St. Louis, MO). The leaves were rinsed twice with sterile deionized water. leaf discs were measured using a flame-sterilized # 3 cork borer cut.

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The were leaf discs aseptically on solid B1 media (step 2) placed. The slices were incubated in the dark at 30 ° C for 4 to 8 weeks, until crumbly were white Callus tissue had proliferated on the leaf disc. the Callus tissue were separated mechanically with forceps and subsequently in 125 ml Erlenmeyer flask transferred (step 3) containing 40 ml liquid B1 media contain. The flasks were located on a rotating shaker at 50 Hz under low-intensive fluorescent lighting (30 μE / m²s) placed. The liquid cultures were after a week with fresh B1 media subcultured.

Two Weeks after the start of liquid cultures Cell clumps were separated using a cell dissociation sieve (Sigma CD-1) with a 60 mesh screen. Approximately two-thirds of the volume the liquid Medium was removed after a cell sedimentation. In step 4, the cells were resuspended in remaining media and 1 ml aliquots were evenly on solid B1 media supplemented with 50 nM Imazetaphyrherbizid (PURSUIT, American Cyanamid, Wayne, NJ) were enriched, streaked ,. The plates were wrapped and matt fluorescent light (5-10 μE / m²s) for 8-12 weeks incubated. <ul><li>c. When imazethapyr concentration of 50 nM, all sensitive cells died. Cell clumps that survived and grew at this concentration are possible as resistant Variants identified. In step 5, these cell clumps were (1-3 mm in transferred diameter) on fresh solid B1 media without herbicides and under low light (10-20 μE / m²s) grown. Each potentially resistant Variant was individually identified and maintained separately.</li><li>d. In step 6, the white, friable callus every 4-6 weeks was on fresh solid B1 media divided to an individual sapling (individual saplings) from the callus growth (en). These shoots were in the following Sucker-cultivation medium (M20) transferred:</li></ul>

Protocol for M20 media

<ul><li>30 g / l sucrose</li><li>100 mg / l myo-inositol</li><li>1.65 g / l NH₄NO₃</li><li>1.90 g / l KNO₃</li><li>0.44 g / l CaCl₂· 2H₂O</li><li>0.37 g / l MgSO₄· 7H₂O</li><li>0.17 g / l KH₂PO₄</li><li>6.2 mg / l H₃BO₃</li><li>16.8 mg / l MnSO₄·H₂O</li><li>10.6 mg / l ZnSO₄· 7H₂O</li><li>0.88 mg / l Kl</li><li>0.25 mg / l Na₂MoO₄· 2H₂O</li><li>0.025 mg / l CuSO₄· 5H₂O</li><li>0.025 mg / l CoCl₂· 6H₂O</li><li>37.3 mg / l Na₂EDTA</li><li>27.8 mg / l FeSO₄· 7H₂O</li><li>1 mg / l thiamine</li><li>0.5 mg / l pyridoxine</li><li>0.5 mg / l nicotinic acid</li></ul>

0.25 mg / l benzylaminopurine

<ul><li>pH 5.95</li></ul>

Celebrations M20 media was autoclaved with 9 g / l and Pflanzenkulturagar with a filter Sterilized herbicide stock solutions as for the selection scheme needed modified. The agar medium in 20 × 100 mm disposable plastic petri dishes cast.

Shoot cultures were 10-20 μE / m²held s fluorescent lighting and by cutting the shoot cultures spread with the blade of a scalpel. Candidate shoot cultures of were expanded and as thereon folgendend on their level of resistance described judged.

2. The degree the imidazolinone resistance in shoot cultures

In Step 7, shoot cultures were judged by their herbicide resistance by three small &lt;?page 6?&gt;Schösslingsausschnitte uniform size on a Plate with M20 media supplemented with logarithmically increasing concentrations enriched in Imazetaphyrherbizid in a range from 1 nM to 0.1 mM is placed. The reaction of the Sir-13 cultures was with those of shoot cultures of a sensitive control (Rel-1) compared. The shoot cultures were for 3 weeks at 25 ° C and at a light intensity of 20 μE / m²s cultured. The reaction of the shoot cultures was an assessment of the violation of the saplings and the fresh weight three weeks after transfer to selective Medium determined. The extent of Herbicide resistance was determined to be sensitive by the ratio of resistant I₅₀Values ​​(the herbicide concentration causing 50% injury).

The Cross resistance characteristics of each variant were in the way determined as described above by the herbicides used in the medium were exchanged. The Schösslingkultur Sir-13 showed a 220-fold level of resistance to imazethapyr and showed no cross resistance to the sulfonylurea herbicide, to chlorsulfuron or the triazolopyrimidine sulfonanilide herbicide to, Flumetsulam as shown in Table 1 below.

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<img img-content="tb" img-format="tif" he="241" wi="103" file="00080001.tif" />

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3. Plant regeneration and growing

In Step 8, shoot cultures were Sir-13 in a similar Way to complete plants regenerated. Two weeks prior to Schösslingssubkulturen Following were Sir-13 shoots transferred into 125 ml Erlenmeyer flasks containing 40 ml N3 Verwurzelungsmedium contain.

Protocol for N3 media

<ul><li>30 g / l sucrose</li><li>100 mg / l myo-inositol</li><li>1.65 g / l NH₄NO₃</li><li>1.90 g / l KNO₃</li><li>0.44 g / l CaCl₂· 2H₂O</li><li>0.37 g / l MgSO₄· 7H₂O</li><li>0.17 g / l KH₂PO₄</li><li>6.2 mg / l H₃BO3</li><li>16.8 mg / l MnSO₄·H₂O</li><li>10.6 mg / l ZnSO₄· 7H₂O</li><li>0.88 mg / l Kl</li><li>0.25 mg / l Na₂MoO₄· 2H₂O</li><li>0.025 mg / l CuSO₄· 5H₂O</li><li>0.025 mg / l CoCl₂· 6H₂O</li><li>37.3 mg / l Na₂EDTA</li><li>27.8 mg / l FeSO₄· 7H₂O</li><li>1 mg / l thiamine</li><li>0.5 mg / l pyridoxine</li><li>0.5 mg / l nicotinic acid</li></ul>

3 mg / l naphthaleneacetic acid

<ul><li>pH 5.95</li></ul>

the N3-medium were dissolved in 40 ml aliquots to 125 ml Erlenmeyer flask divided added 9 g / l agar (0.45 g) was added to each flask and autoclaved for use in rooting of shoot cultures. cultures were then transferred so that it less than 24 hours per day light an average light intensity (40-60 μE / m²s) at 25 ° C were exposed. Roots generally formed 6-8 weeks later.

In Step 9 were, rooted shoots (R₀-Generation) subsequently in a Baccto (Michigan Peat Co., Houston, TX) mixture in -Topf the greenhouse transferred. In step 10 the R were₀plants of Sir-13 crossed with a flachwurzeligen sugar beet called 293. F1 seed from these crosses were self-fertilized to to obtain F2 seeds. It was presumed that imidazolinone resistance a dominant or semi-dominant monogenic trait was. The plants the F2 generation should homozygous at a ratio of 1 resistant 2 heterozygous resistant: 1 homozygous sensitive to imidazolinone resistance auftrenne or mendeln.

Sir-13 Inheritance as a monogenes dominant trait

This relationship was examined by progeny testing each of the F2 plants. In the case of Sir-13, it was granted the F2 plants to fertilize themselves, and seeds were collected separately from each plant. These offspring were in the greenhouse planted and in a two- to four-leaf stage with a ¼ × field application rate sprayed with imazethapyr herbicide (PURSUIT) (0.0625 Ib ai / A + 1 qt / A Sunlt-II (Agsco Fargo) + 1 qt / A 28% UAN (urea ammonium nitrate). These rate can a discrimination of the plants to which one or two copies the Sir-13 allele exhibit of sensitive plants.

Each the Sir-13 families were categorized in homozygous resistant, heterozygous resistant, or homozygous sensitive based on the Distribution of progeny tests. The results clearly showed F2 that the division of the Sir-13 a ratio of 1: 2: 1 for the corresponding Categories corresponds, as is in Table 2 below.

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TABLE 2 <img img-content="tb" img-format="tif" he="36" wi="168" file="00100001.tif" />

These Results suggest that Sir-13 is a monogenic dominant trait is. The degree of dominance or semi-dominance has not to date been determined.

4. imidazolinone resistance whole plants

Homozygous (Non-isolated) Sir-13 F2 plants were self to to produce homozygous Sir-13 F3 seed. Rel-1 clones it was left, to fertilize themselves to produce the sensitive S1 seed. The seeds were in the greenhouse implanted into Baccto-pot mixture and 14 days after planting thinned to one plant per pot. The plants were in four to sechsblättrigem stage sprayed with 0, 0.26, 1.1, 4.4, 17.5, 70 or 280 g active ingredient per ha imazethapyr (PURSUIT) or AC 299,263 (RAPTOR). All treatments were in a volume applied of 239 l / ha and included the following spray additives: 1% per volume Sunlt-II and 1% per volume of 28% UAN. Each treatment was repeated four times. The fresh weight of the shoots was determined 20 days after treatment. The reactions of Sir-13 and sensitive beet are in <figref idrefs="S19">3</figref> shown. The Sir-13 homozygotes each have a 120-fold and 90-fold resistance to Imazethapyr and AC 299,263 on.

5. ALS target enzyme response on imazethapyr

Standard procedures were used to partially ALS from rapidly expanding leaves of greenhouse-grown sugar beet plants purify (Hart, et al Weed Science. 40: 378-383 (1992)). extracts from homozygous plants Sir13- the F3 and Rel-1 S 1 seed were tested for ALS activity in the presence of logarithmically increasing concentrations of imazethapyr. ALS activity was determined from leaf extracts of REL-1 and Sir-13 sugarbeet lines. The plants were in the greenhouse cultured as described above, up to the stage where they four to six leaves exhibit. ALS activity from fresh extracts was determined in the presence of imazethapyr in logarithmically increasing concentrations. The employed Methods and procedures were modified from those were outlined by Ray (Plant Physiol. 75: 827-831 (1984)) and Shaner, et al. (Plant Physiol. 76: 545-546 (1984)). 10 g of sugarbeet leaves were homogenized in 40 ml cold homogenization buffer (0.1 MK₂HPO₄, PH 7.5, 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM Thiamine pyrophosphate, 10 uM Flavin adenine dinucleotide, 10 Vol .-% glycerol) plus 2.5 g polyvinylpolypyrrolidone. The homogenate was filtered through eight layers of cheesecloth and at 27 000 g for centrifuged for 20 minutes. The supernatant was removed and strength to 50% saturation with (NH₄)₂SO₄ brought. These solution at 0 ° C for 1 hour held, then centrifuged at 18,000 g for 15 minutes, the precipitate was then re-dissolved in 1 ml resuspension buffer (0.1 MK₂HPO₄, PH 7.5, 20 mM sodium pyruvate, 0.5 mM MgCl₂) And desalted on a Sephadex G-25 PD-105 column (Pharmacia, Inc., Piscataway, NJ). The enzyme extracts were immediately investigated.

The ALS activity was measured by adding 0.2 ml of enzyme preparation (diluted 3: 1 with resuspension buffer) to 1.3 ml of reaction buffer (25 mM K₂HPO₄, PH 7.0, 0.625 mM MgCl₂, 25 mM sodium pyruvate, 0.625 mM Thiamine pyrophosphate, 1.25 uM incubated flavin adenine dinucleotide) and at 35 ° C for 1 hour. The reaction mixtures contained a final concentration of 0, 4, 40, 400, 4 000, 40 000 400 000 or 4 000 000 nM imazethapyr or 0, 0.9, 9, 90, 900, 9000, 90 000 nM chlorsulfuron. The reaction was stopped by adding 50 .mu.l at 6 NH₂SO₄ and Incubate at 60 ° C for 15 Minutes. This procedure as described by Westerfield (J. Biol. Chem. 16: 495-502 (1945)), decarboxylated and the ALS enzyme product, acetolactate, to form acetoin. A colored acetoin complex was formed by adding 1 ml of 2.5 wt .-% α-naphthol and 0.25 wt .-% of creatine in 2.5 N NaOH and incubating at 60 ° C for 15 minutes.

&lt;?page 10?&gt;

commercially acquired Acetoin was used as a standard for the colorimetric reaction. Acetoin concentrations were determined by measuring the absorbance the reaction solution at 530 nm. Experiments with each herbicide were repeated in each triple execution. Protein concentrations of the extracts were determined by the A method according to Bradford (Anal Biochem 72:.. 248-254 (1976)) using bovine serum albumin for the standard curve.

The Experiments were conducted twice and treatments were repeated three times. <figref idrefs="S20">4</figref> shows the response of ALS extracts Sir-13 homozygotes and the sensitive Rel-1. Sir-13 shows a 150-fold level of resistance to imazethapyr at the enzyme level (as determined by the ratio of I₅₀values determines the resistant and sensitive lines). <figref idrefs="S19">3</figref> shows a 44-fold resistance to the whole plant as discussed above.

6. ALS gene copy number

A Southern blot analysis was performed to determine the number of ALS gene copies in a sensitive sugar beet present to determine. The genomic DNA was isolated from sensitive F3 plants derived from the original Sir-13 mutant, isolated and analyzed using commonly employed techniques (Current Protocols in Molecular Biology, J. Wiley and Sons, New York (1991)). Likewise, genomic DNA from a commercial was Sugar beet varieties analyzed. In both sugar beet lines, was found a single copy of the ALS gene. These dates would show that all ALS enzyme activity of a homozygous sugar beet plant by Mutant-type would consist of the resistant enzyme form.

7. ALS gene mutation

Around the molecular basis for to determine the ALS enzyme resistance, standard techniques were used to sequence two regions of the ALS gene where all previously reported herbicide resistance mutations have occurred (Current Protocols in Molecular Biology, J. Wiley and Sons, New York (1991)). The sugar beet ALS gene had previously been sequenced (U.S. Pat. No. 5,378,824) and Polymerase Chain Reaction (PCR) primers were designed to the two regions of the gene to amplify, which are responsible for previously reported cases of Plant herbicide resistance. A comparison of the sequence information of resistant (Sir-13) and sensitive (Rel-1) sugar beets showed a single nucleotide change from guanine to adenine at position 337 in the nucleotide sequence in a substitution of threonine instead of alanine at position 113 in the sugar beet ALS amino acid sequence results. This site was previously implicated in imidazolinone resistance of burdock (Bernasconi et al, J Biol Chem 270:... 17381-17385 (1995)) and for a sulfonylurea resistance in yeast (US Pat. No. 5,378,824). No other bases changes were for the wild-type nucleotide sequence in the two regions of the examined ALS gene observed.

It is intended that the foregoing description is illustrative only is for the present invention and that the present invention only limited is then on the appended Claims.