Selection process of wheat resistant to herbicides and inhibiting ahas

Wheat resistant to AHAS-inhibiting herbicides and method for its selection

Field of technology

The present invention relates to the use of a screening method for the selection of wheat mutations resistant to acetohydroxyacid α-synthase inhibiting herbicides. The present invention also relates to wheat developed by this method.

Prior art

The first enzymatic step: common in the biosynthesis of branched-chain amino acids (valine, leucine and isoleucine) is catalyzed in plants by an enzyme, acetohydroxyacid synthase (AHAS, known as acetolactate synthase, EC4.1.3.18). AHAS catalyzes two parallel reactions: the condensation of two moles of pyruvate to form acetolactate and the condensation of moles of pyruvate and moles of alpha-keto-butyl. count. This enzyme is inhibited by the end products of the cycle, where condensation produces acetohydroxybutyrate, which is valine, leucine and isoleucine, which is one of the known mechanisms for regulating this cycle in higher plants.

AHAS is a target site for several classes of structurally distinct herbicides; these herbicides include imidazolinones, sulfamoylureas, sulfonylcarboxamides, sulfonamides. _and sulfonylureas.

Large-scale agricultural production is based primarily on the practice of row crops. The main factor enabling f

This practice is the availability of herbicides selectively elimine—

· Herbicides, which control most difficult weeds, are available for most of the main crops grown · The above-mentioned / Ά1Β inhibiting herbocodes are a key element in weed control. However, these herbicides may not affect important weeds in the interstices of the production area. Commonly used herbicides can also cause environmental problems or their use may be limited for cost reasons ·

The essence of the invention

There is a need to develop wheat varieties resistant to JHαS-inhibiting herbicides. The development of such resistant varieties would allow the use of JHjSS-inhibiting herbicides on growing wheat plants, which would result in reduced application rates, reduced groundwater contamination and reduced animal toxicity compared to other groups of herbicides.

Accordingly, it is an object of the present invention to develop a screening method for selecting mutations in wheat resistant to jSHJS inhibition.

In particular, it is an object of the present invention to develop a screening method by selecting imidazolinone-based herbicide-resistant mutations in wheat HHJB-inhibiting wheat.

Another object of the present invention is to identify wheat selection performed by this new screening method.

-3These goals are achieved by cutting the seed, wheat with a chemical mutagen. In the first screening step, the seeds are soaked in a solution containing an AHAS-inhibiting herbicide. In the second stage of screening, after sowing, the seed-containing soil is sprayed with an AHAS-inhibiting herbicide before the plants germinate from the soil. These plants, which germinate in wheat, show resistance to AHAS-inhibiting herbicides.

Brief description of the pictures

Note: Chemical names of protected trademarks and compound numbers: in this brief description of the figures are given in the next part of the description.

Figure 1 shows the effect of increasing the concentration of the imidazolinone herbicide, SCEPTER®¹, for the ratio¹among the number of plants having. normal appearance: and the number of seeds soaked in the solution containing the imidazoline herbicide.

Picture. 2 shows the effect of increasing the concentration of the imidazolinone-based herbicide, PURSUIT, on the ratio between the number of plants having a normal appearance and the number of seeds soaked in a solution containing the imidazolinonone herbicide.

Figure 3 shows the effect of increasing the concentration of the imidazolinone herbicide, ARSENAL, on the ratio between the number of plants having a normal appearance and the number of seeds soaked in a solution containing the imidazolinone herbicide.

Figure 4 and 5 show the appearance of imidazolinone herbicide resistant wheat treated with seed soaking and pre-emergence spray containing PURSUIT®¹ compared to untreated non-resistant wheat. Resistant wheat is in the left part of the area of ​​Figure 4, resistant wheat is in the middle part of the area of ​​Figure 5 ·

Z \ V ^wFigure 6 shows the appearance of wheat derived from seedlings derived from FS4.

Figure 7 shows a comparison of plant heights (in cm) of wild type wheat (BIDEL) against a set of z; PSí four weeks after post-emergence treatment AUTUMN & em ™.

Figure 8 shows a comparison of plant heights (in cm) of wild type wheat / Bidel / versus the FST group four weeks after the post-emergence OUSTenr® treatment.

Figure 9 shows a comparison of plant heights / in cm / of wild type wheat / Bidel / against the set Μ_-Μ. from PS1 four weeks after post-emergence treatment with PURSIUT¹ ·

Figure 10 shows a comparison of plant heights (in cm) of wild type wheat (Fidel) against the M -M set. from PST J en »w four weeks after post-emergence treatment with SCEPTER ·

Figure 10a shows a comparison of plant heights (in cm) of wild type wheat (Bidel) versus the FS1 group four weeks after post-emergence treatment with Compound 2.

Figure 11 shows a comparison of growth (in percent) against control plants / wild-type wheat / Bidel / against wheat M4 mutants derived from BS2 and PS4 týdny four weeks after pre-emergence SCEPTER treatment.

Figure 12 shows a comparison of growth (as a percentage of control plants / wild-type fever) / Fidel / against feverish M4 mutants derived from FS2 and FS4 four weeks after pre-emergence treatment with JJRSENAL ™.

Figure 13 shows a comparison of growth (in percent: against control plants / wild type wheat / Fidel) against wheat M4 mutants derived from FS2 and FS4 four weeks after pre-emergence treatment with PURSUIT ™.

Figure 14 shows a comparison of growth (in percentages) to control plants / wild type wheat (Fidel) to. wheat M4 mutants derived from FS2 and FS4 two weeks after pre-emergence treatment with an imidazolinone herbicide, compound 2.

Figure 15 shows a comparison of growth (in percent against control plants) / wild type (Fidel) against wheat M4 mutants derived from FS2

TO and FS4 two weeks after post-emergence treatment with PURSUIT.

Figure 16 shows a comparison of growth (in percent) against control plants (wild-type fever) (Fidel) against wheat M4 mutants derived from FS2 TO and FS4 four weeks after post-emergence PURSUIT treatment.

Figure 17 shows a comparison of growth (in percent against control plants) of wild-type wheat (Fidel) against wheat M4 mutants derived from FS2 and FS4 four weeks after post-emergence treatment with Compound 2.

Figure 18 shows a comparison of growth (in percent against control plants) of wild-type pelvis (Fidel) against wheat M4 mutants derived from FS2 and FS4 four weeks after post-emergence treatment with Compound 2.

Figure 19 shows a weight / percent comparison of control plants (fresh wild-type wheat plants) (Fidel) against wheat mutants 144 derived from FS2 and FS4 seven weeks after post-emergence treatment with PURSUIT®.

Figure 20 shows a weight / percentage comparison against control plants (fresh wild-type wheat plants (Fidel) against wheat mutants. 144, derived from FS2 and FS4 seven weeks after post-emergence treatment with compound 2 ·

Figure 21 shows a comparison of plant growth (in cm) of wild type wheat (Fidel) against FS2 mutants from a set of 14 -14-seeds four weeks after post-emergence treatment with CLASSIC®.

Figure 22 shows a comparison of plant growth (in cm) of wild-type wheat (Fidel) against FS2 mutants from a set of IIM- seeds four weeks after post-emergence treatment with a sulfonylurea herbicide, OUST®.

Figure 23 shows a comparison of plant growth (in cm) of wild type wheat (Fidel) to FS2 mutants from? four weeks after post-emergence treatment with a sulphamoylurea herbicide, compound 3 ·

Figure 24 shows a comparison of plant growth (in cm) of wild type wheat (Fidel) to FS2 mutants from

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Figure 2g shows a comparison of plant growth (in cm) of wild-type wheat (Fidel) against an FS2 mutant from a seed group four weeks after post-emergence treatment with a sulfonylcarboxamide herbicide, compound 1 ·

Figure 26 shows a comparison of plant growth (in cm) of wild type wheat (Fidel) to the FS2 mutant from the pool ^seaen Four weeks after post-emergence treatment with the imidazolinone herbicide, compound 7 ·

Figure 27 shows a comparison of plant heights (in cm) of wild type wheat (Fidel) versus wheat mutaut. Derived from FS1, FS2 and FS4 three weeks after post-emergence treatment with PURSUIT, compound 2 or imidazolinone herbicide, compound 8.

Figure 28 shows a comparison of plant heights (in cm) of wild type wheat (Fidel) against M.- wheat mutants derived from FS1, FS2 and FS4 three weeks after post-emergence treatment with PURSUIT, compound 2 or compound 8.

Figure 29 shows a yield comparison (in percent versus control plants) of wild-type wheat (Fidel) versus M4 wheat mutants derived from FS1.

FS2a FS4 10 weeks after post-emergence treatment with PURSUIT, Compound 2 or Compound 8.

Figure 30 shows a comparison of growth (in percent against control plants) of wild-type wheat (Fidel) against M4 wheat mutants derived from FS4 6 weeks after post-emergence treatment with PURSUIT®, Compound 2.

-8 mil compound 8, imidazolinone herbicide, CadreTM or sulfonylurea herbicide, JCCENTBa.

Figure 31 shows a comparison of mean area yield (in grams of chi grain perceia / wild type wheat / Fidel) to wheat mutants® FS4 / &amp; 5 seeds / 10 weeks after post-emergence treatment with PURSUIT®, Compound 2, Compound 8, CADREem® or JCCENT®.

Figure 32 shows in vitro enzymatic assays measuring inhibition of ŠHŠS activity in FS1 wheat mutants (designated WS1) not sprayed after germination with herbicide, using valine and leucine (labeled Y + I ») as controls, SCEPTER®, PUESUIT®, compounds 9 and OUSTŮ ™.

Figure 33 shows in vitro enzymatic assays measuring inhibition of šffáS activity in FS1 wheat mutants (designated WS1), sprayed with herbicide® three years old, valihe® and leucine (labeled Y + L) / Mako control, SCEPTER®, PURSUIT®, compound® 9 and UUSTem®.

Figure 34 shows an i® vitro enzymatic assay measuring inhibition of JEEJS activity in wild-type wheat (Fidel), not sprayed after germination with herbicide, using valine and leucine (labeled V + L) as controls, SCEPTER®, PURSUIT®, compound 9 and OUSTem®.

Figure 39 shows in vitro enzymatic assays measuring inhibition of JHJS activity in wild-type wheat (Fidel) sprayed with herbicide® at three weeks of age, valine and leucine (labeled Y + L) as a control, SCEPTER®, PURSUIT®, compound 9 and OUSTem®.

Figure 36 shows an in vitro enzymatic assay measuring inhibition of AHAS activity in ES &lt;wheat mutants, unsprayed, after germination with herbicide, valine and leucine (labeled with V + L) as controls, SCEPTER®, PURSUIT®, Compound 9 and OUST®.

Figure 37 shows an in vitro enzymatic assay measuring inhibition of AHAS activity in FS4 wheat mutants sprayed with herbicide three weeks old, valine and leucine (labeled with V + I ») as controls, SCPTTER®, PURSUIT®, Compound 9 and OUST ™.

Figure 38 shows an in vitro enzymatic assay measuring inhibition of AHAS activity in wild-type wheat (Fidel), not sprayed with herbicide after germination, valine- and leucine (labeled V + L) as controls, SCEPTERea ™, PURSUIT sloučen, compound 9 and OUST ™ ·

Figure 39 shows an in vitro enzymatic assay measuring inhibition of AHAS activity in wild-type wheat (Fidel) sprayed with a three-week-old herbicide, valine and leucine (labeled 7 + L) as controls, SCEPTERea®, PURSUIT®, Compound 9 and OUST ™.

Detailed description of the invention

The present invention is directed to a novel method for screening the selection of wheat mutations having resistance to AHAS-inhibiting herbicides. Wheat seeds are mutagenized by chemical mutagenesis by conventional methods. 7 of the first screening step, the mutagenized seeds are immersed in an AHAS herbicide inhibiting solution using a modified published procedure for wheat soybeans.

In a new, expensive screening step, the seeds are soaked into the soil and sprayed with a herbicide, inhibiting a target of the same class as used in the step preceding the soaking of the seeds before the seedlings germinated from the soil. Wheat plants that have not germinated and have a normal appearance are considered to be resistant to the class of αHαS-inhibiting herbicides used in these two stages. The advantage of these plants, therefore, is that they can be grown on an area that can be treated with a class of JHaS-inhibiting herbicides so as to eliminate difficult weeds while wheat plants remain intact. is demonstrated by testing: several generations of seeds from the original mutants for resistance to a class of herbicides, inhibiting aHA3. The resulting plants may or may not be cross-tolerant to other classes of JEAS-inhibiting herbicides than have been used in the two-step procedure.

Seed mutagenesis is achieved by conventional methods using a chemical mutagen. The procedure described is Kueh and Sfright (Kueh JSH and Bright SWJ ·, Planta, 153, 166-171 (1981)), where the seeds are soaked in water, air is bubbled through them, followed by treatment with a chemical mutagen such as sodium azide. · Seeds are baked. wash with water and dry. The use of sodium azide in this process is particularly advantageous.

Other chemical mutagens and methods for their use include N-methyl-N-nitrosourea and N-ethyl-nitrosourea (Pluhr B.) and Cseplo A., ethods Enzymol. (Plant Mol. Biol.), 118, 611-623 (1986), ethyl methanesulfonate (Sebastian SA, et al., Crop Sci., 29, 14031408 (1989)) and hydroxylamine hydrazine (Khamankar YG, J. Maharashtra TJgrici. Univ. , 14, 322-325 / 1989 // ·

-η

The mutagenized seeds are then examined for their resistance to AHAS-inhibiting herbicides. In vitro tests were originally performed to evaluate herbicide tolerance! for isolated wheat embryos, half-seeds, and whole seeds · However, these tests are time-consuming and problematic due to the problems associated with contamination and variable response in wheat ·^ranother type of selection was used for roto. This procedure is derived from the work developed by Sebastian: and Chaleff / Sebastian. SA and Chaleff RS, Crop Science, 27, 948-952 (1987) for the selection of herbicide tolerant soybeans. This screening procedure involves soaking whole, ripe wheat seeds in a solution containing an AHAS-inhibiting herbicide of a given class of AHAS-inhibiting herbicides for a given period of time, during which the seeds are planted in a sterile soil mixture.

Various classes of AHAS-inhibiting herbicides include imidazolinones, sulfamoylureas, sulfonylcarboxamide, sulfonamides, and sulfonylureas.

Examples of imidazolinone herbicides used in the screening step of soaking include:

2- (4-Isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid, 2- (4-isopropyl) -4-methyl-5-oxo-2-imidazolin-2-yl [3-quinolinecarboxylic acid, 5-ethyl-2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid, 2- (4-isopropyl-4-methyl-5-oxo) -2-imidazolin-2-yl] -5- (methoxymethyl) -nicotinic acid, 5-formyl-2a-4 * isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl] -nicotinic acid, 5- / dimethyl acetal /, 3- (4-propyl-4-methyl-5-oxo-2-imidazolin-2-yl) -2-methylcrotonic acid, 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2 -yl / -5-methylnicotinic acid and a mixture of:

-12&lt;

methyl 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -m-toluate amethyl 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -p-toluate. Use of 5-ethyl-2- (4-isoprepyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid and 2- (4-isopropyl-4-methyl-5-oxo-2-imidazoline -2-yl) -nicotinic acid is preferred. The use of 5-ethyl-2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid is particularly preferred.

Examples of sulfamoylurea herbicides to be used in screening. Seed soaking steps include 1- (4-methoxy-6-methyl-s-triazin-2-yl) -3 - [(propionylphenyl) sulfamoyl] urea, 1- (4,6-dimethoxy)

2-pyrimidinyl [-3- (o-propionylphenyl) sulfamoyl] urea, 1-tert-acetylphenyl] sulfamoyl] -3- (4-methoxy-6-methyl-2-pyrimidinyl) urea and 1 - [[o-Acetylphenyl] sulfamoyl] -3- (4-methoxy-6-methyl-s-triazin-2-yl) -urea. Examples of sulfonylcarboxemide herbicides include 2-acetamido-2,3-dimethyl-β- (p-tolylsulfonyl) butyramide and 2-acetamido-N - (p-chlorophenyl) sulfonyl / -2,3-dimethylbutyramide. An example of a sulfonemide is tert- (2,6-difluorophenyl) -5-methyl-1- (1,2,4) -triazolo [1,5a] pyrimidine-2-sulphon.

phonamide. Examples of the sulfonylurea include 2- (4,6-bis (difluoromethoxy) pyrimidin-2-ylcarbamoylsulfamoyl) benzoic acid, 1- (4,6-dimethoxypyrimidin-2-yl) -3- (dimethoxycarbamoyl-2-pyridylsulfonyl) urea, methyl ester O- [3- (4,6-Dimethyl-pyrimidinyl) -2-yl] -ureido [sulfonyl] -benzoic acid] and ethyl 4- (4-chloro-6-methoxy-2-pyrimidinyl) -carbamoyl (s ulf amoyl) benzoic acid.

If the seeds are soaked for selection, the following procedure shall be used: after drying, the seeds shall be planted and, after a predetermined period of time, such as three or four weeks after planting, the number of germinated seeds shall be counted. The size and appearance of the seedlings are also observed. Seeds,

-13 which germinate and provide seedlings of normal size and appearance may be resistant to JffLAS-inhibiting imidazolinone herbicides.

However, even in the case of seeds, there is a possibility of leakage or false positives from this wet selection: survival after herbicide treatment · To eliminate these false positive results, a preemergent herbicide treatment is used as a second selection.

Gradually soaking the seeds, the semesaa wheat is planted in the soil. If desired, the soil can be irrigated ·

The soil containing the seeds is sprayed with the lever before the seeds germinate from the soil with herbicide® containing: JHáS in aqueous solution or in a solution containing other suitable aqueous or organic solvents. The concentration of the herbicide to be sprayed is determined for each imidazolinone in preliminary tests. on a small scale ·

The JSHJB-inhibiting herbicide to be used in the seed spraying step is selected from the same class as the herbicide used in the seed soaking step. The selected herbicide may be the same or different in this two-step selection, but in each In this case, these herbicides must be selected from the same class of HHAS-inhibiting herbicides.

The seeds subjected to this two-stage selection are then. longer evaluated after germination of seedlings from the soil. Growth in normal size, yield and appearance indicates that wheat includes mutations having the desired resistance to inhibiting herbicides. JHlflS.

—14'

Wheat with a high level of tolerance to αELAS-inhibiting herbicides is selected by this two-step screening. This screening system allows for the preferential selection of highly resistant wheat plants by seed exposure, herbicide doses, lethal to wild-type wheat in two-stage selection, seed soaking and pre-emergence application. This double exposure of the herbicide eliminates false positives and thus serves to eliminate weakly resistant mutants. These resistant wheat plants are resistant to the herbicides of the class used in the two-step selection. Hosts may in certain cases be cross-tolerant to one or more other classes of JH4S-inhibiting herbicides, but this cross-tolerance may not be present.

The examples below illustrate the present invention on the imidazolinone class of inhibiting herbicides. JKáS.

Other classes of JH4S-inhibiting herbicides are also within the scope of this invention, as their mechanism of action is the same as that of imidazolinones. All herbicides that inhibit 4H4S have the same mechanism of action acting on the ŠHJS enzyme, thereby blocking the biosynthesis of valine, leucine and isoleucine: By blocking the activity of ŠHJS, these herbicides inhibit the metabolism of plants, leading to their death.

If the two-step screening method according to the invention is used with the imidazolinone class of herbicides, a selection of wheat is obtained which is resistant to the imidazolinone class of herbicides. Herbicide-resistant wheat has an increased tolerance to imidazolinones of up to 50 times. compared to non-mutagenized unselected wheat. Imidazolinones have been shown to have an inhibitory effect on the enzyme SHAS, one of the first enzymes in amino acid biosynthesis; with branched chain, selected wheat

-15 show the activity of AHAS that is not inhibited by imidazolinones as determined by the in vitro enzymatic assay (described in the examples below). Unfortunately, there is little or no evidence of cross-tolerance to other chemicals with a similar mechanism of action on AHAS in terms of plants or enzymatic studies.

From the 117,000 seeds of wheat that were screened, four types of wheat plants were selected for their resistance. The selections of these four mutants are designated PS1, PS2, PS3 and FS4. Seed samples of these wheat plants were deposited March 28, 1991 at Aaerican Type: Culture Collection, 12301 Parklawn Drive, Hockville, MD 20852 and were identified by the following reference numbers:

mutant wheat storage number

PSI JECC 40994

PS2 ATCC 40995

PS3 ATCC 40996

PS4 ATCC 40997. Based on genetic data, the selected wheat mutants PSI, PS2, PS3 and PS4 are allelic and appear to be the result of a single mutational effect. Tolerance is inherited as a single genetic locus and is dominant or semi-dominant in expression. Increased herbicide tolerance has been shown to have no detrimental effect on grain yield, both in the presence and absence of the imidazolinone herbicides tested.

The inheritance of mutations in resistant wheat selections is confirmed by testing several generations of seed progeny, as well as hybrids of mutant selection. the test seeds are subjected to both pre-emergence and post-emergence

-16 application of various classes of herbicides inhibiting AHAS. The size, yield and appearance of the seedlings are observed in comparison with untreated control plants (Fidel) and control plants (Fidel) subjected to the same two-stage screening procedure.

/

The results of these assays, as described in the examples, indicate that mutations having resistance to imidazolinones are inherited from generation to generation of plants derived from selections FS1, FS2, FS3 and FS4. The specificity of the resistance is demonstrated by data indicating that mutant selections are not resistant to: other classes of AHAS-inhibiting herbicides such as sulfonylureas, sulfonoureas, sulfonamides or sulfonoylcarboxamides.

In a second embodiment of the invention, two different classes of inhibitory herbicides are used in a two-step screening procedure. AHAS. A herbicide of one class is used in the seed soaking step. A second class herbicide is used in the spraying step. Selections of plants that survive and grow normally are resistant to both classes of herbicides: Although the frequency of mutations having resistance and their selection will be greatly reduced due to the different classes of herbicides used, if the number of selected seeds is large enough, selections with double resistance.

The following examples are provided to better understand the present invention. The examples are illustrative only and are not to be construed as limiting the scope of the invention.

-17Examples of the invention

Development of two-stage seed selection *

I. Seed mutagenesis

Five thousand seeds of French winter wheat Fidel ** shall be mutagenised in accordance with ^£ueha a ^ righta / Kueh

JSH and Bright SWJ, Planta, 153, 166-171 (1981). The wheat seeds are soaked in water for 18 hours at 0 DEG C. and bubbled with air for 6 hours at 20 DEG C., followed immediately by treatment with 1 mM sodium, chemical mutagen, at pH 3 for two hours. Wash with water for 30 minutes, and then spread in a thin layer on a paper towel to dry. After drying, the seeds are planted in the field.

Wheat® plants are allowed to grow until development is complete and M2 seeds are harvested. About 117,000 wheat seeds are harvested and used to select imidazolinone-resistant wheat. the germination frequency of this material is 100 #.

2 · Degree. 11. Seed soaking

By the procedure developed by Sebastian and ^ haleff s®, wheat seeds are surface-thickened for 30 seconds in 70 #

EtOH, followed by dehumidification in 50 # solution, contains TM clm 2,625 # sodium hypochlorite and 1 to 2 drops of Tween 20 (a nonionic surfactant, polyethylene sorbitan monaurate), available under the registered trademark of Atlas Chemical Industries, in 100 ml of solution for 30 minutes, under vacuum with gentle stirring using a mixing plate. The seeds are washed in sterile distilled water and the lever is soaked in the herbicide solution for a specified time as indicated in the mouse. A set of twenty-five seeds is

Place the 18pek in 25 ml of the herbicide solution below in a sterile plastic petri dish and leave to soak for the specified time. The seeds are then washed with water, dried with a paper towel and sown (25 seeds / area) about 1/2 ”deep into a 6x8 inch peat container containing moist sterile Metro Mix 350 ™ soil /^Grace: Company, Cambridge, MJ / · Treatment results are evaluated four weeks after treatment. The herbicides, their doses and duration of action are listed below:

Experiment 1:

AUTUMN JL ™ í 0, 1, 10, 50, 100, 500 / μM

SCEPTER ™: 0, 1, 10, 50, 100, 500 / UM for 48 hours. Each treatment is repeated four times. / JESEŇJL ™ and SCEPTER ™ are trademarks of Anerican Cyanmr ámiďl Company. ARSEN is 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid and is described in U.S. Patent No. 4,798,619, SCEPTER ™ is 2- (4-isopropyl) -4-methyl -5-oxo-2-imidazolin-2-yl] -8-quinolinecarboxylic acid and is described in U.S. Patent No. 4,798,619.

ARSENJL Inhibits seed germination and seedling growth with increasing concentration - although at concentrations up to 50 .mu.m, some false positive results can be visually observed, higher concentrations completely inhibit seedling growth. At these concentrations of 100 and 500 / UM, some susceptible seeds germinate, but the seedlings are small, twisted and generally abnormal in appearance.

Although SCEPTER ™ also inhibits seedling growth. with increasing concentration, three to four weeks after germination, this phenomenon compensates, as can be seen visually. In fact, seedlings treated with 1-100 yuM can not? distinguish from control plants and seedlings treated with 500 μM have slightly lower growth than control plants.

Pokue 2

ARSEN AL ™ 0, 10, 50, 100, 500 / uM

TM

SCEPTER® 0, 100, 250, 500, 750, 1000 / UM for 48 hours. Each experiment is performed twice.

Tm

ARSENAL was tested again at the same concentrations. as in the previous experiment and the same results were obtained as. is described above. In this experiment, the increase was increased

Sena concentration-SCEPTER at maximum, 1000 μM. At the end of this experiment, it was clear from visual observation that the concentration was still not high enough to prevent leakage. " The duration of herbicide exposure also appears to affect the number of false-positive findings. Some examples of root elongation occurred when the seeds were soaked for several hours longer, however, in this experiment, the germinates did not appear at all after the treatment with the herbicide treatment. * The effect of seed soaking time on the herbicide concentration required to prevent leakage is evaluated in the following experiment.

Experiment 3

<td>ARSEN AL ™</td><td>O,</td><td> 50,</td><td> 75,</td><td> 100</td><td>/ UM</td><td></td><td></td>

<td>TM SCEPTER</td><td>O,</td><td> 250,</td><td> 500,</td><td> 750,</td><td> 1000,</td><td> 1500</td><td>/ UM</td>

<td>PURSUIT ™</td><td>O,</td><td> 250,</td><td> 500,</td><td> 750,</td><td> 1000,</td><td> 1500</td><td>/ UJÍ</td>

compound 10, 500, 1000, 1500, 2000 / UM

-20 after 48, 72 or 96 hours. Each treatment is repeated three times / PURSUIT® is a trademark of the American Yanamid Company. PURSUIT ™ is 5-ethyl-2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid and is described in U.S. Patent δ »4798619 · Compound 1 is 2-acetamido-N- (p-chlorophenyl) sulfonyl] -2,3-dimethylbutyramide and is described in U.S. Pat. 4992094 / · In this experiment, the imidazolinone herbicides ARSEN AL®, SCEPTER®, PURSUIT® and sulfonylcarboxamide Compound 1 were evaluated as selective agents in seed soaking selection, and the interaction between seed soaking time and herbicide concentration was evaluated. In Figures 1 to 3 are. results of treatment with imidazolinone herbicides are given. If the seeds are soaked.48 hours, the number of normal / plants decreases with increasing herbicide concentration. As the duration of herbicide exposure increases, the concentration of herbicide required for lethality decreases. ^Rro ARSENAL ™ is a sufficient concentration: 50 / ul if the exposure is greater than 72 hours, however 75 / taM is necessary if the duration of exposure is shorter. SCEPTER is not effective at any herbicide concentrations / exposure duration, whereas treatment of 1000 / ul PURSUIT cells for 72 hours or 750 ul for 96 hours prevents the survival of possible false positives. Compound 1 has no inhibitory effect on either seed germination or seedling growth.

Experiment 4

SCEPTER ™ 0, 500, 1000, 1500, 2000 / UM for 3, 4, 7 and 10 days. Each treatment is repeated twice.

Use the procedure described above, except that 3550 seeds are soaked in a petri dish. 25 seeds are again planted in the container. This experiment was repeated twice. / Note: In the first experiment, day 7 to 10, the seedling was contaminated with mold and destroyed. In the second experiment, a fungicide, Captan, was added to the soaking solution to prevent contamination.

Soaking times of seven and ten days are too long to use with wheat seeds. At this time, it begins to appear with SCEPTER®¹ elongation of roots at concentrations; up to Bo 1000 μM; during these time periods, there are also problems with mold contamination.

The visual results are similar to those found for PURSUIT in Experiment 3. For mutant selection. imidazolinone-resistant concentrations are suitable concentrations of SCEPTERut bu; 1000 or 1500 / μM for 3 days to minimize the problem of false positive results.

3 · Stage 2: Freemergent herbicide application

A series of experiments were performed to determine the appropriate amount of herbicide for this pre-emergence spray application. Wheat seeds of a suitable Fidel cultivar are sown in 6x8 inch peat containers filled with Metro Mix 350®® at a rate of 100 seeds, per container. Containers are irrigated before herbicide treatment ·

During the treatment, three containers are sprayed, the herbicides are applied with a laboratory belt sprayer at a rate of 950 liters per hectare (l / ha) at a belt speed of 12.8 s.ot ~ * using a spray nozzle No. 40015E (Téejet®® Spraying Systems). . ? In three to four weeks will be done

-22visual evaluation of treated plants in terms of toxicity to wheat / seedling seeds. PURSUI® and SCEPTER® are most effective at a rate of 300 to 350 g / ha, while

TM

J5RSENJL is most effective at pre-emergence spraying 40 to 50 g / ha.

4 «Screening and selection of herbicide resistant mutants.

PURSUIT was used as an example of imidazolinone to screen for herbicide resistance. Mg wheat seeds were surface de-stained for 30 seconds, after which; followed by deaeration in a $ 50 solution containing $ 2.625 sodium hypochlorite as described above. The seeds were then washed three times with sterile distilled water and placed in a sterile 100 x 15 mm plastic petri dish, 250 seeds per dish. Twenty-five ml of a 1000 μM solution of PURSUIT was added to each bowl of levers and the seeds were left soaked in this solution in the dark for 3 days. The seeds were dehydrated and dried on a paper towel and then sown in sterile 6x8 inch peat containers containing Metro Mix, 1000 sown seeds / container. The containers were weighed and immediately sprayed with 300 g per hectare / g / ha / of PURSUIT®. The herbicide was applied with a laboratory belt sprayer at a rate of 950 l / ha at a belt speed of 12.8 s.ot¹ using spray nozzle No. 400152 · After four weeks, the seedlings were evaluated for herbicide tolerance. Screening of wheat seeds took place gradually in sets of 25,000 seeds. From about 117,000 seeds, wheat that underwent a two-stage herbicide resistance selection, four wheat plants resistant to PURSUIT® were selected.

23These plants are striking in appearance compared to non-resistant foams. Two of these mutational selections are shown in the figures. 4 and 5. Resistant wheat is in the leftmost chamber in Figure 4, resistant wheat is in the middle container of Figure 5. These four, mutation selections are designated FS1, PS2, PS3 and FS4 * Resistant plants were transplanted into 7.5 inch peat pots to Metro Mix 350.

Examples 2-5

Characterization of herbicide resistant mutants

Attach 2

Heredity of herbicide resistance

1. screen plantsJ in vivo

M1 plants from the four original plant selections (designated PS1j_PS2, PS3 and PS4) were evaluated for PURSUIT resistance. Five Mn seeds from each mature plant of these original mutants were dewashed in 70% ethanol for 30 seconds, followed by deworming in a 50% solution containing 2.625% sodium hypochlorite (as described above) for 20 minutes under vacuum with gentle agitation. using a mixing plate · The seeds were then washed twice in sterile distilled water.

Each set of five seeds was then placed in 10 ml of a 1000 / UM solution of PURSUIT ™ in a sterile 60 x 15 mm plastic Retri dish and soaked for three days. · The seeds were then washed with water, dried with a paper towel and sown in 6x8 peat containers. inches, approximately 1/2 deep into sterile Meters of Mix 350® * soil. These containers were pre-emergently sprayed with PURSUITBm at a rate of 300 g / ha. Control samples contained Mn seeds from each resistant selection, which were delaminated as described above, soaked in sterile distilled water, and then sown. In addition, the unselected seeds of Fidel susceptible wheat were discarded as described above, one half of which was treated with PURSUIT (seed dipping and spraying) and the other half was soaked in water alone. The herbicide was applied with a laboratory belt sprayer at a rate of 950 l / ha at a belt speed of 12.5 s.ot ~ 1 using a spray nozzle No. 4001533 · four weeks after the initial soaking of the seeds, the wheat plants were evaluated for herbicide. resistant, damaged by the herbicide or killed. The herbicide-resistant plants were then transplanted individually into 7.5-inch azalea pots containing mg

Metro Mix 350. One week after transplanting, the plants were fertilized to a leaf with Osmocote fertilizer (Sierra Company, Milpita, CA). In addition, the plants were regularly fertilized with Peter® s 20-20-20 fertilizer (Grace Company, Cambridge, MA). Individual plants were evaluated against untreated control plants Pidel ·

Memutagenized, unselected seeds did not survive treatment with PURSUIT ™ at a concentration of 1500 χθΜ / seed soaking / and 300 g / hs (pre-emergence application). The same seeds, when soaked in distilled water and without spraying, germinate within 1-2 weeks with an average germination of 63.3% (average of two sets of control plants). Seeds M ^. When soaked in sterile distilled water and without spraying had a germination of 53.3 Seeds of resistant selections of wheat treated with PURSUIT ™ had the following germination: PS1-60 PS2-47.5 fc, PS3-41.2% and FS4-55 % · “Leg of seedlings

The 25 treated herbicides have the same growth rate as the untreated control plants, however, some FS4-derived seedlings are affected by the herbicide, have stunted growth, are twisted and / or have chlorotic bands (Figure 6). ⁱⁿMost of them are * capable of survival and further growth. The height of the plants three weeks after the herbicide treatment is given in Table 1.

Table 1: Plant screen: results of progeny screening of four original wheat selections when evaluated by seed dipping / pre-emergence application.

Selek- number number average height range of more ears of plants ^ shoot / cm / hunts / cm /

<td>FS1</td><td> 16</td><td> 48/80</td><td> 9,1</td><td> 2-15</td>

<td>FS2</td><td> 16</td><td> 38/80</td><td> 11,7</td><td> 5-18</td>

<td>FS3</td><td> 17</td><td> 35/85</td><td> 8,0</td><td> 1-14,5</td>

<td>FS4</td><td> 1?</td><td> 44/85</td><td> 21,1</td><td> 5-28</td>

<td>control</td><td> 1 —²</td><td> 6/15</td><td> 19,0</td><td> 10-26</td>

<td>control</td><td> 2 -²</td><td> 13/15</td><td> 21,2</td><td> 8-30</td>

<td colspan="2">control 3</td><td> 8/15</td><td> 28,9</td><td> 27-31</td>

¹Number of emerged plants / number of planted.

²Control samples 1 and 2 are the number of seeds rather than the ears.

Control 1 = sensitive wheat seeds (Fidel) soaked in sterile distilled water.

Control 2 »sensitive wheat seeds (Fidel) soaked in herbicide solution. Control of seeds from the original selections, soaked in sterile water.

The height of the plants of the nautical selections is in the range of 1 to 28 cm. This indicates variability in the degree of herbicide tolerance. Control plants had plant yields ranging from 8 to 31 cm. All plants were transplanted into 71/2 inch Raelin pots in Metro Mix 350 soil and used for the crossing described in Part 4 of this Example 2 (Genetic Atavine Study).

2. plant screen in vitro Fiostils from the four original plant selections (designated FS1, PS2, FS3 and PS4) were evaluated in vitro for resistance to Pursuit ™. The progeny seeds of each mature plant of these Kg mutants were degreased in 7OS6 ethanol for 30 seconds, followed by decontamination in 5056 solution containing 2.625% sodium hypochlorite (as described above) for a period of time. 20 minutes under vacuum and gentle stirring using a mixing plate. The seeds are then. soaked in sterile distilled water in a 100 ml and 15 mm plastic sterile petroleum dish for 20 to 24 hours, after which the embryos were removed and cultured in a medium containing either 10 .mu.l, 10 .mu.m or 10 .mu.l of PURSUIT. Control samples contained non-mutagenized embryos (Pidel) cultured on herbicide-free and herbicide-treated media and samples on herbicide-free medium. Embryo evaluations were performed either two and a half weeks or 3 weeks after the start of culture.

None of the embryos of the control samples or cultures cultured in medium containing 10 PUBSUIT germinated, although certain embryos showed a small elongation of the primary loop roots (1 to 5 mm). TO₃ embryos are slightly more tolerant to PURSUIT ™ in the medium at a concentration of 10 ~ 5m, however, significant growth inhibition persists. Table 2 shows the embryo ratings where the "T" is tolerable,

-27D "damaged, S" sensitive effect on embryos cultured in medium containing 10 "⁵M a 10% PURSUIT ™.

Table 2: Plant screen: Results of progeny screening of four original wheat samples in in vitro seed evaluation. Evaluation of embryos -T- tolerant, D^M-damaging, ^MS- sensitive.

PURSUIT ™ concentration »10⁵

<td rowspan="2">Selection</td><td colspan="3">evaluation of edtyí</td><td rowspan="2">average height of shoots / cm /</td><td rowspan="2">shoot height range / cm /</td>

<td>T</td><td>D</td><td>WITH</td>

<td>PS1</td><td> 21.</td><td> 16</td><td> 42</td><td> — —</td><td>⁰ - 1 ,5</td>

<td>PS2</td><td> 21</td><td> 14</td><td> 45</td><td> —-</td><td> 0 - 3,5</td>

<td>PS3</td><td> 12</td><td> 9</td><td> 58</td><td> —-</td><td> 0 - 4,5</td>

<td>FS4</td><td> 16</td><td> 15</td><td> 31</td><td> -—</td><td> 0 - 3,0</td>

<td rowspan="2">control 1 control</td><td> 10</td><td> 0</td><td> 0</td><td> 15,6</td><td>í - 28</td>

<td rowspan="2"> 0</td><td rowspan="2"> 0</td><td rowspan="2"> 30</td><td rowspan="2"></td><td rowspan="2"></td>

<td> 2</td>

<td>control</td><td> 27</td><td> 1</td><td> 1</td><td> 14,7</td><td> 1 - 28</td>

PURSUIT ™ concentration = 10⁶

<td colspan="4">Embryo evaluation selection</td><td rowspan="2">average height of shoots / cm /</td><td rowspan="2">shoot height range / cm /</td>

<td colspan="2">Τ '</td><td>D</td><td>WITH</td>

<td>PS1</td><td> 70</td><td> 0</td><td> 0</td><td> 9,1</td><td> 1 - 22</td>

<td>PS2</td><td> 64</td><td> 1</td><td> 0</td><td> 11,4</td><td> 1 - 23</td>

<td>PS3</td><td> 34</td><td> 12</td><td> 22</td><td> 5,2</td><td> 1 - 23</td>

<td>PS4</td><td> 59</td><td> 8</td><td>T3</td><td> 13,4</td><td> 1 - 33</td>

<td rowspan="2">control 1 control</td><td> 20</td><td> 0</td><td> 0</td><td> 23,1</td><td> 14 - 32</td>

<td rowspan="2"> 0</td><td rowspan="2"> 0</td><td rowspan="2"> 15</td><td rowspan="2"></td><td rowspan="2"></td>

<td> 2</td>

<td>control</td><td> 15</td><td> 0</td><td> 0</td><td> 14,6</td><td> 2-28</td>

-28Control 1 = sensitive wheat seeds (Fidel) on herbicide-free media. Control 2 = sensitive wheat seeds (Fidel) on herbicide medium. Control of M ^ s seeds from the original selections on herbicide medium.

Based on these in vitro results, mutant selections show a 10- to 100-fold increased tolerance to RURSUIT ™.

3. Segregation studies

The study was performed to determine resistance to imldazolinones and the homogeneity of the progeny of the original selections of wheat mutants. They were used in this study. seeds from plants / derived, from original selections. FS1-FS4 / who survived the two-stage screen described above. In addition, seeds derived from plants of each selection that were not included in the selection (designated as storage plants) as well as Fidel control plants were evaluated. Seeds derived from both the stock and® screened plants representing the result of each original selection of wheat (FS1-FS4) were sown in containers at twenty-five seeds per row and eight rows per container. One week after sowing, at the Z12 seedling stage (two leaves), the plants were sprayed with FURSUIT® at a rate of 62.5 g / ha.^HThe ebicide was applied with a laboratory belt sprayer at a rate of 950 l / ha at a belt speed of 12.5 s / l * 1 using a No. 40015E spray nozzle 18 inches above the plants. Twéen 20® was used as a surfactant at a concentration of 0.25 # v / v. The plants were evaluated three weeks after spraying.

Fidel. Samples are completely sensitive to application

62.5 fe / ha of PURSUIT ™ / all plants perished /. Imid azblinone-resistant selections of winter wheat showed excellent

-29 tolerance to post-emergence application of PURSUIT ™ at a dose of 62.5 g / ha in this greenhouse assessment. Progeny derived from selections FS1, FS2 and FS4 showed homogeneously resistant to PURSUIT®®, although several susceptible plants and offspring with low susceptibility to germination were observed. These three selections appear to be homozygous resistant. The z.FS3 progeny may be sensitive, split, or may be completely resistant. Progeny with divided characteristics with good germination have about one quarter of susceptible individuals. This selection is apparently heterozygous and requires further self-generation to obtain a uniform homozygous material. Sowings to increase the number of seeds are carried out in the fields. Following this test in the greenhouse, the progeny with indivisible characteristics are collected from each individual selection so that a sufficient number of seeds are available for the field tolerance test / field test 1, part 1 of example 4 / ·

4. Genetic status studies

To determine whether the selections of mutants. wheat are. allelic. / and perhaps derived from the same mutation / or different, mutants need to be crossed. It is also necessary to cross resistant types with sensitive types (Fidel) to determine the inherited properties of the resistance phenomenon. From this point of view, all possible crossings and their reciprocal implementation between the parent lines Fidel, FS1,

FS2, FS3 and FS4 · The F 1 hybrids that emerged from these crosses were evaluated for resistance, selfing and were back-tested by crossing: with susceptible individuals to produce a herd to determine heredity and information on hellism for the duration of selection.

-30Pofcus 1: Six chambers containing Metro Six 350®® were seeded with P hybrids derived from the following crosses: PS1 χ Pidel, PS2 x Pidel, PS3 x Pidel, PS4 x Pidel and Pidel (sensitive basic cultivar). After one week, the chambers were sprayed, each with a dose of herbicide, PURSHFIT®®, at a dose of 50, 100, 150, 200 and 250 g / ha to determine the genetic status of the trait of the herbicide resistance property of PS1-PS4 selection. the herbicide was applied with a laboratory belt sprayer at a rate of 950 l / ha at a belt speed of 12.5 s.ot ^ using a spray nozzle No. 40015B · ^ Tween 20 at a concentration of 0.25% · 2 was used as a surfactant in the TO solutions of the herbicide.

Plants were evaluated 10 days after herbicide treatment.

These hybrids are growth restricted but are not killed at 250 g / ha of PURSUIT®®, indicating that even in heterozygous conditions these plants show relatively high levels of imidazolinone resistance. Pidel plants are killed at a rate of 150 g / ha of PURSUIT®® and are severely damaged at rates of 50 and 100 g / ha. These hybrids, PSI x Pidel, PS2 x Pidel, PS3 x Pidel and PS4 x Pidel all show herbicide resistance in the P1 generation. A segregation ratio of 1 resistance: 1 susceptible individual could indicate a heterozygous condition. However, this was not observed in any of the four selections, and based on these data, the gene carrying herbicide resistance in wheat resistant mutants is dominant and homozygous.

Experiment 2: This experiment was performed to evaluate selections of wheat mutants? due to their herbicide resistance to cross-breeding and selfing. Cross-breeding was performed between M 1 plants representing selections from the original four mutants and between these selections and the Bidel type. The progeny of these hybrids, as well as the self-pollination of the plants used in the crossing, are sown in asphalt azalea pots in Metro soil.

-31Mix 350 ™ * Ten pots for each crossing are sown with two m and seeds per pot · Ten days later, all offspring, self-pollinated and reciprocally crossed, were sprayed with PURSUIT ™ at a rate of 200 g / ha. Herbicide treatment was applied; procedure in experiment 1 · The number of plants was then limited: per plant per pot / by removing weakly from two plants from the pot / one week after herbicide application · Germination of seeds of mutant cross-breeding mutants was 100% and more than 90% in seed mutants originating from from self-pollination. No cross-mutant specimen was damaged by the PURSUIT ™ challenge.

Experiment 3rd Hybrids Fj. hybrids (described in Experiment 1) and Pidel type are sprayed with a dose of 200 g / ha of PURSUIT ™.

The following hybrids of Metro-Mix 350 půdy are sown in 6-inch azalea pots. The number of planted flower pots for one crossing is indicated by the number after the crossing:

Pidel x / Fidel / FSl / 12

Pidel x. / Fidel / FS2 / 211

Pidel x / Fidel / FS3 / 9

Pidel x / Pidel / PS4 / 8

Fidel? 20 / * 7 each of the twenty pots are two plants of the Pidel type /. About a month later, the plants are sprayed (except for the Pidel type). PURSUIT ™ / 200 g / ha / was applied as described above. The plants were evaluated three and a half weeks later.

Of the 49 progeny tested that were sprayed, 29 survived and 20 were killed · The 29:20 distribution is close to the 1: 1 distribution expected for dominant / or semi-dominant / genetically controlled inheritance · However, the putative heterozygotes in this experiment were significantly more damaged than plants sprayed with the same dose of herbicide in the previous experiment · It is not entirely clear whether treatment at a later stage of growth causes increased damage to heterozygotes or whether other factors are present.

Experiment 4: Resistant selections of FS1-FS4 wheat were crossed with type. Fidel and seeds F ^. of these crosses were subjected to selfing. The crossings were as follows:

/ Fidel x FS1 /, / Fidel x FS2 /, / Fidel x FS3 /, / Fidel x FS4 /, self-pollination self-pollination self-pollination

F₂ the offspring of these crosses and their susceptible mother variety Fidel were sown in chambers. Five F1 progeny were used for each of the four crosses. One chamber was sown with each progeny from each cross. In the chambers there were jritt six rows in each, in a row there were 25 seeds. The chambers were then sprayed post-emergence eleven days later with PURSUIT® at a rate of 200 g / ha. PURSUIT ™ was applied as described above.

-33This dose of PURSUIT ™ proved to be lethal to the susceptible type of wheat, while it proved to be non-lethal to heterozygous or homozygous resistant types. Segregation for herbicide resistance in this progeny confirmed that the resistance property for each of the Four selections (FS1-FS4) is hereditary as the only dominant or semi-dominant property. The Fg progeny of each of the four crosses provide an apparent 3: 1 ratio (resistant / susceptible) in post-emergence: PURSUIT ™ spraying at a dose of 200 g / ha.

Experiment 5: Elala crosses and reciprocal crosses of four mutant wheat selections were performed. The seeds of Fj from these crosses were sown and the plants were left to self-pollinate. Fg seeds were harvested and used in this experiment to determine the allelism of these four selections. The chambers were filled with ^ etro 350 ^ soil and sown with 100 seeds from one cross (four rows of 25 seeds per row). In addition, one row of sensitive Fidel wheat was sown in each chamber. Three sources were used from each crossing. Upon reaching the Z12 stage seedlings, the chambers were sprayed with PUHSUIT® at a rate of 200 g / ha. PURSUIT solution ^⁴ was applied as previously described, four weeks after treatment, the number of resistant individuals was determined relative to the number of susceptible plants.

Studies of allelism between the four resistant selections of wheat show that all selections are allelic or closely related, with few or no susceptible susceptible plants found between crosses between selections in the Fg generation. Even if the genes are non-allelic and linked, the progeny are thought to divide at a rate of 15 (resistant): 1 (susceptible). Table 3 shows the results of these genetic conditions.

34Table 3: Genetic condition studies: Number of plants that are resistant / sensitive to 200 g / ha of PURSUIT®. Data refer to three weeks after treatment.

<td colspan="2">Resource resistant</td><td colspan="3">perception source resistant</td><td>susceptible</td>

<td>/ FS1 / FS2 / S-2</td><td> 71</td><td> 1</td><td>/ FS2 / FS1 / X-1</td><td> 95</td><td> 0</td>

<td>/ FS1 / FS2 / X-2</td><td> 85</td><td> 1</td><td>/ PS2 / FS1 / X-2</td><td> 91</td><td> 0</td>

<td>/ FS1 / BS2 / X-3</td><td> 74</td><td>t</td><td>/ FS2 / FS1 / X-3</td><td> 97</td><td> 0</td>

<td>total</td><td> 230</td><td> 4</td><td>total</td><td> 283</td><td> 0</td>

<td>/ FS1 / FS3 / X-1</td><td> 82</td><td> 0</td><td>/ FS3 / FS1 / X-2</td><td> 95</td><td> 0</td>

<td>/ FS1 / PS3 / X-2</td><td> 92</td><td> 0</td><td>/ FS3 / FS1 / X-3</td><td> 97</td><td> 0</td>

<td>/ FS1 / FS3 / X-3</td><td> 93</td><td> 0</td><td>/ PS3 / FS1 / X-5</td><td> 94</td><td>T</td>

<td>total</td><td> 267</td><td> 0</td><td>total</td><td> 286</td><td>t</td>

<td>/ FS1 / PS4 / X-1</td><td> 96</td><td> 0</td><td>/ FS4 / FS1 / X-1</td><td> 93</td><td> 1</td>

<td>/ FS1 / FS4 / X-2</td><td> 90</td><td> 0</td><td>/ FS4 / FS1 / X-2</td><td> 91</td><td> 0</td>

<td>/ FS1 / FS4 / X-3</td><td> 93</td><td> 2</td><td>/ FS4 / FS1 / X-3</td><td> 94</td><td> 1</td>

<td>total</td><td> 279</td><td> 2</td><td>total:</td><td> 278</td><td> 2</td>

<td>/ FS2 / FS3 / X-1</td><td> 99</td><td> 0</td><td>/ FS3 / FS2 / X-1</td><td> 96</td><td> 0</td>

<td>/ FS2 / FS3 / X-2</td><td> 87</td><td> 1</td><td>/ BS3 / FS2 / X-2</td><td> 94</td><td> 0</td>

<td>/ FS2 / FS3 / X-3</td><td> 92</td><td> 0</td><td>/ FS3 / FS2 / X-3</td><td> 96</td><td> 0</td>

<td>total</td><td> 278</td><td> 1</td><td>total</td><td> 286</td><td> 0</td>

<td>/ FS2 / FS4 / X-1</td><td> 102</td><td> 0</td><td>/ FS4 / FS2 / X-1</td><td> 94</td><td> 0</td>

<td>/ FS2 / FS4 / X-2</td><td> 97</td><td> 0</td><td>/ FS4 / FS2 / X-2</td><td> 95</td><td> 1</td>

<td>/ FS2 / FS4 / X-3</td><td> 97</td><td> 1</td><td>/ FS4 / FS2 / X-3</td><td> 95</td><td> 0</td>

<td>total</td><td> 296</td><td> 1</td><td>total</td><td> 284</td><td> 1</td>

<td>ZFS3 / FS4 / X-1</td><td> 98</td><td> 0</td><td>/ FS4 / FS3 / X-1</td><td> 96</td><td> 0</td>

<td>/ FS3 / FS4 / X-2</td><td> 96</td><td> 0</td><td>/ FS4 / FS3 / X-2</td><td> 100</td><td> 0</td>

<td>/ FS3 / FS4 / X-3</td><td> 96</td><td> 0</td><td>/ FS4 / FS3 / X-3</td><td> 94</td><td> 0</td>

<td>total</td><td> 290</td><td> 0</td><td>total</td><td> 290</td><td> 0</td>

35In no case is the number of susceptible segregants approaching 1/16 of the total number / Table 3 /. No maternal effects on heredity were also observed. These data support the probability that all four selections are derived from or very closely related to the same mutation event.

Experiment 6: Seeds of self-pollinated progeny from the second backcrossing of Fidel-type resistant mutants (susceptible progenitor cultivar) were sown in standard chambers filled with Metro Mix 350 ™ soil. Thirty seeds were sown on one source. Seven to ten days after TM sowing, these chambers were sprayed with PURSUIT at a rate of 200 g / ha as described above. Three weeks after treatment, the plants were evaluated for segregation of resistance properties. As expected, a certain part of the offspring is completely resistant, the other part is divided in terms of resistance. Some offspring are resistant and some offspring are susceptible, while another offspring is fully susceptible to the herbicide. Homozygous resistant plants are selected and used for seed propagation (in the nursery) to obtain a resistant cultivar of improved properties.

Example 3

Herbicide resistance studies · level and spectrum of herbicide resistance

In this greenhouse test, the level and spectrum of herbicide resistance of mutaut selections at the level of the whole cultivar was evaluated, four imidazolinones (PURSUIT ™, SCEPTER ™, ARSEN AL ™ and compound 2) and one sulfonylurea / OUST ™, registered trademark EIdu Pont De were tested. Nemours and Company, which is · N - [3- (4,6-dimethylpyrimidin-2-yl) ureido] -36 sulfonyl] benzoic acid methyl ester. Compound 2 is a potentially short residual imidazolinone and is 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -5- (methoxymethyl) -nicotinic acid. The growth rate, determined by plant height, is compared with untreated controls. Seeds derived from plants that have been screened for herbicide resistance using two-stage selection (seed soaking and spraying) and from unscreened stock plants were collected after being. proven non-segregating property / see segregation studies / · These lines were collected according to their parent lines: FS1, FS2, FS3 and FS4. As a control sample. and the Fidel type (wild type) was used for comparison purposes. Five seeds from each of these lines (including the Fidel type) were sown separately in six-inch azalea pots, and later their number was limited to three pot plants. Three determinations per line and six determinations for wheat lines used as untreated controls were used per treatment (type of chemical treatment and dose). The plants were sprayed post-emergence ten days after sowing with the following herbicides in the following doses:

imidazolinones:

<td>pursuit ™</td><td> 20, 40, 80, 160, 320</td><td>g / ha</td>

<td>SCEPTER ™</td><td> 40, 80, 160, 320, 640</td><td>g / ha</td>

<td>ARSEN AL ™ *</td><td> 3, 6, 12, 24, 48</td><td>g / ha</td>

<td>compound</td><td> 2 20, 40, 80, 160, 320</td><td>g / ha</td>

<td colspan="2">sulfony urea · OUST ™ 5, 10, 20, 40, 80</td><td>g / ha</td>

-37 / * Note: Plants sprayed with JRSENJL ™ are sown three days earlier than plants treated with other methods, however the evaluation is performed simultaneously with other treatments · In addition, although three trials of single doses of JRSENJL ™ were evaluated, mostly two plants instead of three / · It was added to the herbicide solutions before spraying

Tween 20 ™ at a rate of 0.25% v / v was added to the herbicide solutions before spraying. The herbicides were applied with a laboratory belt sprayer at a rate of 400 l / ha at a height of 18 inches above the wheat plants at a belt speed of 8.2 s.ot1. using No. 65015E spray nozzle. Plant heights are measured immediately before spraying and levers at one-week intervals for four weeks.

The four selections are similar both in terms of their spectrum and in terms of their level of resistance. The PS3 type is slightly less resistant than the other three selections. Because the PS3 type was selected as heterozygous, heterozygous or susceptible seeds may be inadvertently included in these experiments. Based on plant growth four weeks after treatment, the mutant wheat was found to be resistant to a dose of more than 320 g / ha PURSUIT, more than 640 g / ha SCEPTER ™, more than. 48 g / ha JRSENJLu ™, more than 80 g / ha of compound 2 and more than 5 g / ha of OUST ™ · • These values ​​can be demonstrated for an approximately 8-fold increase in tolerance to PURSUIT ™, more than a 16-fold increase in tolerance to SCEPTER ™ and JBSENJLu ™, a 16-fold increase in tolerance to compound 2 and a 2-fold increase in tolerance to OUST ™. Figures 7-10a show the tolerance of one wheat selection mutant (PS1) to the respective herbicides, JRSENAL®, OUST®, PURSUIT®, SCEPTER® and Compound 2.

—38—

2. Pre-emergence herbicide tolerance

TM

In this experiment, the imidazolinones PURSUIT, ARSEN JL, compound 2 and sulfonylurea - pre-emergence (in contrast to the post-emergence application described in Part 1 of Example 3) were applied to the Pidel wheat variety and herbicide-resistant PS2 and PS4 selection to assess their herbicide tolerance.

Seeds of two mutants of PS2 and PS4 selections and seeds susceptible to the Pidel variety were used in this experiment.

Five seeds of each type of wheat were sown in six-inch azalea pots on Sassafras soil / sandy soil / · Four parallel experiments for each wheat type and six parallel experiments for each wheat type in untreated controls were used for one treatment type and dose. samples. The day after sowing the seeds, the pots were sprayed with the following

<td colspan="6">bicides at the indicated doses:</td>

<td>PURSUIT ™</td><td> 40,</td><td> 80,</td><td> 160,</td><td> 320, 640</td><td>g / ha</td>

<td>Sceptejr ™</td><td> 40,</td><td> 80,</td><td> 160,</td><td> 320, 640</td><td>g / ha</td>

<td>Arsenal ™</td><td> 2,5,</td><td> 5,</td><td> 10,</td><td> 20, 40</td><td>g / ha</td>

<td>compound 2</td><td> 5,</td><td>it,</td><td> 20,</td><td> 40, 80</td><td>g / ha</td>

<td>oust ™</td><td> 5,</td><td> 10,</td><td> 20,</td><td> 40, 80</td><td>g / ha</td>

The herbicides were applied as described above. / Level and spectrum of herbicide resistance / · After spraying, the pots were irrigated. Plant height was measured four weeks after treatment.

-39Based on plant growth after four weeks, FS2 and FS4 selections were found to have approximately B-fold increased tolerance to ARSENAL ™ and PURSUIT ™ / fig.

and 13) and 8 to 16-fold increased tolerance to compound 2 (FIG. 14 / and SCEFTER ™ / fig. 11 /. The increase in TM tolerance to OUST appears to be 2 to 4-fold. Similar results were observed for root and shoot gains (weight) determined seven weeks after treatment.

3 · Post-emergence herbicide tolerance

In this second experiment, in the greenhouse tulle evaluated the upper limits of resistance to post-emergence application of PUS_TM

SUIT of α-compound 2. Seeds of PS2 and PS4 mutant selections and Fldel-type seeds were used in this experiment. Five. seeds of each type were sown in one six-inch escalator pot in; sterile soils Metro Mix 350 ™. Immediately before herbicide treatment, the number of plants was reduced to three plants per pot. Three parallel assays were used per treatment (herbicide type and dose) for each wheat type and six parallel assays for untreated controls.

The plants were then treated by spraying post-emergence 10 days after sowing with the following herbicides and doses (g / ha): PURSUIT ™: 10, 25, 50, 100, 250, 500, 1000, 2500 compound 2: 2.5, 5, 10, 25 , 50, 100, 250, 500 herbicides were applied as already described. The height of the plants was measured immediately before spraying.

Plant height was also measured two and four weeks after treatment and shoot weight gain was measured one week after treatment.

-40Dv® weeks after treatment in® all combinations showed selections of delayed symptoms compared to untreated controls / fig. 15 to 17 / · However, four weeks after treatment, the heights of FS2 and FS4 plants were the same as in untreated control plants at doses. 500 to 1000 g / ha of PURSUIT ™ (FIG. 16) and 25 to 50 g / ha of compound 2 (FIG. 18 /. Said selections show a 40- to 100-fold increase in tolerance to PURSUIT and Compound 2 applied post-emergence, in this test compared to the type. Fidel / unselected /. Data relating to the weight gain of the shoots seven weeks after treatment show that S2 and FS4 have a weight gain of shoots equal to 90 to 100% with respect to the growths of the control plants (Figures 19 and 20).

4 · Pre-emergence tolerance to sulfamoylurea to determine if selection of imidazolinone-resistant wheat. also indicate resistance to sulfamoylurea herbicides, two experiments were performed. Resistance to. four sulfamoylurea herbicides (described below) and one sulfamourea herbicide (OUST®) / were tested in a post-emergence and pre-emergence test ·

In the first experiment, seeds of selected FS2 wheat mutants and Fidel-type seeds were sown in six-inch azalea pots in Sassafras soil / sandy soil / · Initially, three seeds were sown per pot, then the plants were limited to two. yljc performed: three parallel determinations for each treatment. In addition, fifteen parallel experiments with untreated plants were used for both the Fidel and FS2 types. The plants were sprayed pre-emergently with the following herbicides and doses.

41Sulfamoylureas:

<td>compound</td><td> 3</td><td> 12,5,</td><td> 25,</td><td> 50,</td><td> 100,</td><td> 200</td><td>g / ha</td>

<td>compound</td><td> 4</td><td> 62,5,</td><td> 125,</td><td> 250,</td><td> 500,</td><td> 1000</td><td>g / ha</td>

<td>compound</td><td> 5</td><td> 12,5,</td><td> 25,</td><td> 50,</td><td> 100,</td><td> 200</td><td>g / ha</td>

<td>compound</td><td> 6</td><td> 12,5,</td><td> 25,</td><td> 50,</td><td> 100,</td><td> 200</td><td>g / ha</td>

Compound 3 is 1- (4-methoxy-6-methyl-s-triazin-2-yl) -3- (o-propionylphenyl) sulfamoyl] urea, compound 4 is 1- (4,6-dimethoxy-2-pyrimidinyl) -3 - [o-propionylphenyl] sulfamoyl] urea, compound 5 is 1- (acetylphenyl) sulfamoyl] -3- (4-methoxy-6-methyl-2-pyrimidinyl) urea and compound 6 is 1- / (o-Acetylphenyl) sulfonyl-3- (4-methoxy-6-methyl-s-triazin-2-yl) urea. All four compounds are described in U.S. Patent No. 4,622,565.

Sulphonylureas and:

OUST ™ 1.56, 3.125, 6.25, 12.5, 25 g / ha

Herbicides were applied as described previously.

Very low emergence was observed in the pre-emergence test and again no differences were observed between Fidel type and resistant selections.

5. Post-emergence tolerance to sulfamoylurea

In the second experiment, M 1 -M 2 seeds from FS2 mutant wheat selection and Fidel-type seeds were sown in six-pale azalea pots in sterile soil "etro Mix 350". Originally, three seeds were sown on a pot, gradually the plants were reduced to two. Three parallel determinations were used for each type of treatment. Moreover

-42, 15 parallel experiments of untreated control plants of both Fidel and PS2 types were included. The plants were sprayed post-emergence, 12 days after sowing with the following herbicides in? listed doses:

Sulfamoylureas: compound 3 12.5, compound 4 82.5, compound 5 12.5, compound 6 12.5, sulfonylurea: OUST ™ 6.25,

25, 50, 100, 200 g / ha 125, 250, 500, 1000 g / ha 25, 50, 100, 200 g / ha 25, 50, 100, 200 g / ha

12.5, 25, 50, 100 g / ha

Herbicides were applied as previously described.

Resistant and susceptible wheat was treated with doses of herbicides that were thought to cause significant damage to lethalization of susceptible wheat types. Unfortunately, the susceptible cultivar of wheat / Fidel / was not affected / in the visual, evaluation / not even the highest! doses of sulfamoylurea herbicides in a post-emergence test. Is it good? Fidel type wheat relatively tolerant to sulfoboyl-based herbicides, or? the doses are not high enough to cause damage, or the herbicide does not cause damage for any other reason (formulation, environmental impact, etc.) The resistant wheat type shows a 2-4-fold increase in OUST tolerance in this experiment. These results are similar to those obtained in previous experiments.

-436. Wheat herbicide resistance spectrum M 1 -M 2.

In this experiment, the tolerance of imidazolinone-resistant wheat to three sulfonylurea herbicides / BEACON ™, CLáSSIC ™ and OUST ™ / BEACON ™ is a registered trademark of Ciba-Geigy and is 2- / 4,6-bis = / difluoro methoxy / -pyrimidine-2. -ylcarbamoylsulfamoyl / benzoic

TM acid, CLASSIC is a registered trademark of Ε.I. du Pont de Nemours and Company and is ethyl 0- (// 4c chloro-6-methoxy-2-pyrimidinyl) -carbamoyl / sulfamoyl (benzoic acid), sulfamoylurea herbocod (compound 3), sulfonylcarboxamide (compound 1). / and an imidazolinone herbicide / compound 7, which is

3- (4-Isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -2-methyl-crotonic acid to detect cross-tolerance to AHAS-inhibiting herbicides other than imidazolinones. The seeds of M ^ -M ^ mut ant selection of PS2 wheat and Pidel type seeds were sown in five-inch azalea pots in sterile Metro-Mix 350 půdy soil. Originally, three seeds were sown on a pot, later the number of plants was limited to two. Each treatment experiment consisted of three parallel determinations. In addition, six parallel determinations of both FS2 and Pidel types were used as control untreated plants. Plants were sprayed post-emergence: seven days after sowing with the following herbicides and doses *

Sulfonylureas *

BEACON ™ 15.6, 31.3, 62.5, 125, 250 g / ha

CLASSIC ™ 31.3, 62.5, 125, 250, 500 g / ha

OUST ™ 6.25, 12.5, 25, 50, 100 g / ha

Sulphamoylureas:

compound 3 125, 250, 500, 1000, 2000 g / ha

Sulfonylcarboxamide:

Compound 1,250, 500, 1000, 2000, 4000 g / ha

Imidazolinones:

Compound 7: 187.5, 375, 750 g / ha

Herbicides were applied as described above, and the plants were evaluated for the second, third and fourth weeks after application of the herbicide.

Cross-resistance to sulfamoylureas (Fig. 21, 22 and 24), sulfamoylurea (Fig. 23) or sulfonylcarboxamide

Xobr.25 / is 0 e &amp; 2-fold and is similar to the resistance poww observed in previous experiments with OUST. Resistance to imidazolinone compound 7 is similar to that previously observed for PURSUIT® or JRSENal® (greater than 10-fold). No appreciable increase in resistance to non-imidazolinone herbicides was observed.

Example 4 - Field tests

1.Field experiment I

A field trial was performed to evaluate the tolerance of imidazolinone-resistant wheat selections PS1-PS4 to post-emergence imidazolinone herbicides. The Pidel variety from which these selections are derived is included as a control. Because Pidel is a French winter wheat variety unsuitable for New Jersey, this test was designed to evaluate the relative effects of hernicidal treatments on crop yields rather than on the absolute yield of individual genotypes.^rroto yields are also reported as

-45 grams / box instead of bushels / acre or t / ha.

Collected M seeds were used in this field experiment₄ parent lines PS1, FS2, FS3 and FS4 and the original Fidel type. The test was performed on an incompletely divided field with a main area for herbicide treatment and a separate area for genotypes. Three experiments were performed for each treatment (per dose and type of chemical), but for mutant wheat selection, no determinations No. 2 and No. 3 were performed simultaneously due to the small number of collected seeds. The fields are approximately three feet long and 1.5 meters wide (7 rows with 7 inch row spacing) with about 25 seeds per row sown. ^Mthere is a meter gap between the individual wheat lines and a ten-meter gap between parallel experiments .

... The following treatments were performed: untreated controls

<td>PURSUIT ™</td><td> 100</td><td>and</td><td> 200</td><td>g / ha</td>

<td>compound 2</td><td> 50</td><td>and</td><td> 100</td><td>g / ha</td>

<td>compound 8</td><td> 100</td><td>and</td><td> 200</td><td>g / ha</td>

Compound 8 is 5-formyl-2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -nicotinic acid -5- (dimethylacetal) and is described in published European Patent Application No. 322616. .

The treatment was carried out at a rate of 400 1 / ha with a sprayer connected to a tractor. Tween 20 ™ was used as a surfactant. The wheat was treated at the Z22 stage or at a height of approximately 6 inches. Plant height and degree of damage were assessed at 3 and 6 weeks after treatment. Yields were evaluated 10 weeks after treatment.

Three weeks after treatment, resistant wheat selection does not appear to be affected by herbicide treatments when evaluating plant height. 27 /. No clearly visible effects of herbicide treatment were observed. In the untreated state, these selections were slightly shorter compared to the untreated / Fidel type. The susceptible Fidel cultivar was severely stunted as a result of these treatments.

Six weeks after treatment, the height of plants resistant to wheat selections was still unaffected by herbicide treatment / FIG. 28 /. The Fidel type was severely stunted or the plants died as a result of herbicide treatment compared to resistant selections, which are clearly unaffected by these treatments. The final height of the untreated plants was almost the same as the height of the untreated Fidel plants, although at three weeks after selection treatment they were lower than the untreated Fidel plants.^ofResistant wheat selection is thought to grow somewhat more slowly than untreated Fidel-type plants, probably due to undesirable recessive plants. mutations. These recessive mutations can be removed by backcrossing methods known to those skilled in the art.

Types FS1, FS2 and FS4 show no reduction in yields after post-emergence treatment with any of the above bicecides. In contrast, the Fidel type shows 3596 reductions in yield and more than 35% when treated with PURSUIT. 7556 yield reduction with other herbicides, treated Fidel plants had significantly higher yields than FS1 or FS4 (respective yields 133 g / field, 40 g / field and 76 g / field). The mean yield of the FS2 type (98 g (field)) was thus lower than the yield of the untreated Fidel type, but was not significantly lower at the tenth week after treatment (FIG. 29 /. Lower

The yields of these selections are not unexpected, the authors show less vitality and naturalization of the plant probably due to the presence of undesired genes causing mutagenesis but not related to the resistance property. imidazolinone. Somewhat surprisingly, the FS1, PS2 and PS4 types show such large differences in yields considering that all three types are most likely derived from the same mutational stimulus · These selections appear to be completely resistant to herbicide treatment both in terms of plant height so in terms of grain yields.

2.Field field H

A second field study was performed to evaluate the tolerance of imidazolinone-resistant PS4 wheat selection to the post-emergence application of imidazolinone herbicides - PURSUIT ™, CADRE ™, compound 2 and compound 8 and urea herbicide sulfones * - JCCBNT ™. CADRE ™ is a registered trademark of Anerican Cyanamid Company. CADRE is 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -5-ethylnicotinic acid and is described in U.S. Patent No. 4,798,619. JCCENT is a registered trademark of EIdu Pont de Nemours and Company and is 1- (4,6-dimethoxypyrimidin-2-yl) -3- (3-dimethylcarbamoyl-2-pyridylsulfonyl) urea. The Ridel type was included as a susceptible control variety.

Seeds from the PS4 lines of the highest yield in field test I were collected and used in this field test together with seeds of the Pidel type. The test was performed on an incompletely divided field with a main area for herbicide treatment and a separate area for genotypes. Eleven treatments were performed with three replicates for each treatment (type of chemical and dose). The fields were three meters long and 1.5 meters wide

-48Imidazolinones: PURSUIT¹100 and 200 compound 2 50 and 100 compound 8 100 and 200

CADRE ™ 50 and 100 sulphonylureas: JCCEÍT¹“20 and 40/7 rows with 7 inch row spacing / with a seedling density of approximately 100 seeds / m. A gap of 0.5 meters was maintained between the wheat lines and a gap of three meters between other determinations »

The following treatments were applied: untreated control plants g / ha g / ha g / ha g / ha g / ha

The treatment was carried out at a rate of 200 l / ha by means of a back-sprayer. were approximately 30-35 cm high. Evaluation of the height of plants and their degree; the damage was done

1st, 2nd, and 3rd · and 6th week after treatment. Yields were evaluated 10 weeks after treatment.

After three weeks, the growth of Fidel plants was found to be severely inhibited by all types of herbicide treatments. Resistant FS4 selections show tolerance to all of the imidazolinones tested, however, no cross-resistance was observed to the sulfonylurea herbicide (AJCENT®). Giant. 30 shows evaluations performed in the sixth week after treatment, indicating that the post-emergence treatment of PUR-49TM TM

SUIT. , CADREm, compound 2 or compound 8 has no effect on the final height of the plant, but this resistant wheat has little or no tolerability to

TM w

ACCENT · The comparative susceptible variety hardly includes or dies at these doses, herbicides. Grain yields expressed as the average of three determinations are shown in Figure 31. · The grain yield of resistant wheat FS4 selection is not reduced in any of the imidazolinone herbicide treatments. Fidel yields are severely limited or eliminated by imidazolinone treatment. herbicides. Both the resistant types and the susceptible type of Fidel provide only. little or no grain yield after herbicide treatment: ACCENT ™. The effects resulting from Fidel type imidazolinone treatments are. far worse than the plant height reduction described above.

The resistant wheat used in this field test was obtained by collecting several yields harvested in the field I test. ^Nand each additional field was sown with seeds from one plant, although no backcrossing was performed with this type of material to the Fidel type, seed sources, with low germination and agronomic efficiency could be ruled out. You_withand_EThe yields of resistant selections were much improved compared to the Field I experiment. The yields obtained for resistant types for untreated control plants were the same as for untreated plants of the susceptible Fidel type. This indicates that the inclusion of an imidazolinone resistance gene has no effect on grain yield.

Example 5

Enzyme study

Initial determination of the plant /

Herbicide application: plants originating from four

-50 native mutant plants (FS1, FS2, FS3 and FS4) were tested for the metabolic activity of AHAS (acetohydroxyacid synthase). Plants showing resistance after soaking the seeds and screen after spraying with PURSUIT ™ were transferred from the chambers to individual pots. After reaching three years of plant age, two plants of each FS1-FS4 mutant selection retained additional PURSUIT ™ at a rate of 62.5 g / ha, while two plants from each selection remained untreated. Treated and untreated Fldel-susceptible plants were also included in this experiment. Approximately one-half of the foliage above the soil line from each plant is removed two days after herbicide application and the inhibition of AHAS by valine and leucine (as a control) is determined

SCEPTER®, PURSUIT®, OUST® and a sulfonylcarboxamide herbicide, Compound 9 · Compound 9 is 2-acetamido-2,3-dimethyl-N- (p-tolylsulfonyl) butyramide and is?

described in U.S. Patent No. 4,883,914 ·

Slight resistance to PURSUIT-resistant wheat types was evident in unsprayed plants. However, a significant increase in TM AHAS resistance to PURSUIT is seen with FS1-FS4 spraying compared to spray.

unbred type. Figures 32-35 show the results for. j is one of the four resistant selections (FS1) and for the unbred / susceptible / Fidel type. From these results it appears that the resistance of FS1-FS4 to PURSUIT ™ is due to the conversion of TM

AHAS, which is resistant to inhibition by PURSUIT ·

Enzyme extraction: AHAS extraction was performed on 10 graa tissue in liquid nitrogen and homogenized in 100 mM potassium phosphate buffer (pH 7.5) containing 10 mM pyruvate, 5 mM MgCl 2, 5 μM EDTA. 100 / UM FAD,

-511 mM valine, 1 mM leucine, 10% glycerin and 10 mM cysteine. The homogenate was then filtered through a nylon cloth (53 μM mesh) and the lever centrifuged at 25,000 g for 20 minutes. Supernatant. was brought to 50% saturation with (HH 2) 2 SO 4 and allowed to stand on ice for 20-30 minutes. If centrifuged at 25,000 g for 20 minutes, the supernatant was discarded. Ammonium sulfate pellets were dissolved in 50 mM potassium phosphate buffer (pH 7.5) containing: 1 mM EDTA and 100 mM NaCl and the solution was used. to determine the content.

Determination of AHAS: The potency of AHAS was measured by evaluating the product, acetolactate, after conversion by decarboxylation in the presence of acid to acetic acid. Standard reaction mixtures contained. enzyme in 50 mM potassium phosphate buffer (ρΗ * 7.0) containing 100 mM sodium pyruvate, .10 mM MgCl 2, .1 mM thiamine pyrophosphate and 10 .mu.M FAD. The mixture was incubated at 37 ° C for one hour and then quenched by the addition of sulfuric acid to a final concentration; in the tube was 0.85% HgSQ2. The reaction product is allowed to decarboxylate at -60 ° C for 15 minutes. The acetoinase formed is determined by incubation with creatihent (0.17%) and 1-naphtholea (1.7% solution in 4® NaOH) according to Westerfield (Westerfield WW, Biol.). Chem., 161, 495-502 (1945) · The maximum color is reached after incubation at 60 DEG C. for 15 minutes with further incubation at room temperature for 15 minutes. of the color complex is measured at 520 nm. Appropriate verification of the direct formation of acetoin during the enzymatic assay is performed. Each determination shall be performed at least in duplicate and the experiment repeated at least twice.

-522. Enzyme assay I / M ^ plants /

-Herbicide application: Two chambers were sown with approximately 100 seeds from the selection of foam (FS1, FS2 and FS4) and FideL type into sterile Metro Mix 350 soil. After ten days, each chamber was sprayed postemergently

PURSUIT in an amount of 62.5 g / ha. The spray rate was 400 l / ha at a belt speed of 8.2 s.ot ^ using a nozzle δ. 65015E. Tween 20 at a concentration of 0.25% v / v was used as a surfactant. The plants were harvested three days later and the effectiveness of AHAS was determined.

Enzyme extraction and AHAS assays were performed as described above.

3 · Enzyme assays II / M ^ plants /

Herbicide Application: It was performed as described above (Enzyme Assay 1) except that the spray rate was 950 L / ha at a belt speed of 12.8: s.ot -1 * 1 using Nozzle No. 4G015E.

Enzyme extraction and AHAS assay: was performed as described above.

The results of these two experiments were very similar, the efficacy of AHAS in all four homozygous mutant lines of unsprayed plants is slightly resistant to PURSUIT srovnání compared to the wild-type enzyme &amp; control plants (Fidel). However, significantly higher levels of UR resistance to PURSUIT were observed for the enzyme from the selections that were sprayed compared to the enzyme from the sprayed Fidel plants / Fig. 36-39 /. She was too

-53Some resistance to SCEPTER ™ and OUST ™ was observed, but in * PK / f the level of resistance was lower than for PURSUIT. Spectrum and level; resistance to different herbicides is very similar in all four mutants, suggesting that all four mutants resulted from a single mutagenic stimulus. This conclusion is confirmed by the genetic experiments described above.