Subject of Invention

The present invention refers to a chemical way of exploiting a heterosis in commercially significant hermaphrodite plant species, preferably from the family of grasses (lat. Poaceae), especially common wheat (lat. Triticum aestivum L.), in which easily soluble or water-soluble derivatives of oxanilic acid of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof or water-soluble preparations that contain them are used as an active chemical hybridization substance.

The invention further refers to a method for the production of hybrid seeds of Fi generation (the first filial generation) of commercially significant hermaphrodite plant species, especially common wheat, with chemical hybridization comprising:

selection of seeds of both parent components so that sowing of seeds of both parent components is possible in the form of separate strips or optionally in the form of a mixture of mutually mixed seeds of both parent components,

wherein in the case of sowing a mixture of seeds the female component of a hybrid variety (line AA) expresses a natural or genetically engineered resistance against a herbicide, preferably a non-selective herbicide, and the male component of a hybrid variety (line BB) does not express such resistance and wherein in the case of sowing the seeds of both parent components in the form of a mixture inflorescence of a pollinator (line BB) occurs when the end of the premeiotic stage occurs in the female component of the hybrid variety (line AA) due to flowering synchronisation of both parent components and due to prevention of induction of a high percentage of male sterility in the pollinator, induction of male sterility in the plants of the female component of the hybrid variety (line AA), wherein easily soluble derivatives of oxanilic acid of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof are applied onto plants from the end of the premeiotic stage of the plants of the female component on and/or when the latter are in the meiotic stage, and optionally in the case of sowing seeds in the form of a mixture of both parent components, after the completed fertilisation of the plants of the female component (line AA), removing of the plants of the male component from the crop by applying a herbicide, preferably a non-selective herbicide, against which the plants of the female component express resistance, whereas the plants of the male component or pollinator do not have this resistance, onto plants and their locus.

In the case of sowing a mixture of mutually mixed seeds of both parent components of a hybrid variety the method of the invention provides for a simpler design of a seed crop for the production of hybrid seed, better spatial exploitation, better pollination of the female component (line AA) and a greater quantity of seeds of the desired F, generation based on the sown quantity of both parent components in comparison with the approaches used hitherto.

Prior Art and Technical Problem

The invention refers to a method for the exploitation of a heterosis in plants with hermaphrodite florets (hereinafter referred to as hermaphrodites or hermaphrodite plant species), especially in common wheat, with a chemical induction of male sterility. Heterosis presents superiority of the first filial generation (F] generation) over the parental generation, which is expressed for instance in a higher yield, lower mycotoxin content, more rational consumption of plant nutrients etc. For the exploitation of heterosis hybrids produced by a controlled cross between two genetically different homozygous parental components are used (cross $ line AA x $ line BB) (Blouet, A., Streiff, ., Guckert, A. (1999): Possibilities for hybrid seed production in wheat. V: Heterosis and hybrid seed production in agronomic crops. Basra A.S. (ed.). Binghamton, Food Products Press ^® : 81-108.)

In order to produce a hybrid seed of plant species with hermaphrodite florets an efficient system for inducing male sterility is needed. Induction of male sterility means that only the female sex starts expressing on a hermaphrodite plant species.

In the past, several genetic and transgenic approaches were suggested for the induction of male sterility in hermaphrodite plant species. Common to the systems for the induction of male sterility based on cytoplasmic-genetic male sterility, the transgene for the synthesis of cytotoxic or cytostatic polypeptides and the transgene for the formation of an exogenous double- stranded RNA for the induction of RNA interference, is the preservation of male-sterile female component with its fertile analogue (three-component system). Unlike genetic and transgenic approaches to the cultivation of hybrid variety based on three distinct lines ($ line AA, isogenous line A ^' A ^' , S line BB) the chemical induction of male sterility needs only two parent components of the hybrid variety (two-component system). In addition to this, the advantage of chemical induction of male sterility is also the absence of complex genetic engineering and long-term input of a male- sterile cytoplasm with backcrossing (Wirmani et al. (2003): Two-line Hybrid Rice Breeding Manual. Hardy B. (ed.). Metro Manila, IRRI).

A relation between a selective effect of a chemical on the metabolism of a plant in the sense of induction of male sterility (gametocidic activity) and production of a hybrid seed could be traced as far back as 1957. In that year, the Method for the production of a hybrid cotton seed (lat. Gossipium hirsutum L.) by using the agent FW-450 (produced by Rohm & Haas) was described. An established term for the chemicals having a distinctive selective effect on microsporogenesis or development of viable pollen and with which chemicals it is possible to produce a hybrid seed is a chemical hybridizing agent (abbreviated CHA) (McRae, D.H. (1985): Advances in chemical hybridization. Plant breed. Review, 3: 169-191).

Chemical hybridizing agents are efficient in inducing male sterility in the female component of the hybrid variety; however, their application in more general production of a hybrid seed is often too complicated due to a need to separate both parent components.

Current approaches to a chemical exploitation of heterosis require sowing of both parent components in the form of strips. When producing a hybrid seed of common wheat (lat. Triticum aestivum L.) which is a typical hermaphrodite plant species, the strips of a female component (line AA) are normally 6 to 20 m wide and the pollinator's strips (line BB) are 1.5 to 6 m wide. Sowing of parent components in the form of strips is very demanding in practice and what's more, the central part of the strip of the female component is often poorly pollinated due to a huge distance from the location of pollen occurrence (Cisar, G., Cooper, D.B. (2002): Hybrid wheat. V: Bread wheat. Curtis B.C., Rajaram S., Gomez Macpherson H. (ed.). Rome, FAO Plant Production and Protection Series).

An important problem in chemical exploitation of heterosis is also a selection of the active substance, since most of the chemical hybridizing agents express a strong phytotoxic effect in the early stages of organogenesis (Blouet, A., Streiff, K., Guckert, A. (1999): Possibilities for hybrid seed production in wheat. V: Heterosis and hybrid seed production in agronomic crops. Basra A.S. (ed.). Binghamton, Food Products Press ^® : 81-108).

Consequently, the parent components cannot be sown in the form of a mixture due to flowering synchronisation, wherein the pollinator must lag behind in development. In the group of chemicals inhibiting microsporogenesis the representatives from the group of oxopyridazines and oxy- and amino-substituted cinnolines have become widely accepted as commercial agents for the chemical hybridization of common wheat (Dotlacil, L., Apltauerova, M. (1978): Pollen sterility induced by ethrel and its utilization in hybridization of wheat. Euphytica, 27: 353-360; Graham, R.D., (1986): Induction of male sterility in wheat using organic ligands with high specificity for binding copper. Euphytica, 35: 621-629; Wong, M. et al. (1995): Effectiveness of SC2053 as a chemical hybridizing agent for winter wheat. Plant Growth Regulation, 16: 243-248).

Known representatives of oxopyridazines with gametocidic activity are fenridazon (1- (4-chlorophenyl)- 1 ,4-dihydro-6-methyl-4-oxopyridazine-3-carboxylic acid) and clofencet (2-(4-chlorophenyl)-3-ethyl-2,5-dihydro-5-oxopyridazine-4-ca rboxylic acid). In 1970s and 1980s the company Rohm & Haas developed a series of oxopyridazines (RH-531 , RH-532, RH-2956, RH-4667, RH-5148, RH-0007), among which the active substance fenridazon (RH-0007) reached a commercial value in the form of the agent Hybrex ^® . Clofencet in the form of the agent GENESIS ^® (also MON 21200) was registered in 1997 by the company Monsanto, yet it needed to be withdrawn from use after several years due to environmental unacceptability. In order to achieve an efficient induction of male sterility the agent GENESIS ^® (22.4 %) needs to be applied between the phenophases EC 32 and EC 39 (visible ligule of a flag leaf) in a dosage ranging from 1.4 kg to 2.7 kg of active substance per hectare. The pollen grains of plants treated with the active substance clofencet have a wavy surface and the exine is by 80 % thinner. The pollen grains are incapable of germination due to plasmolysis (United States - Environmental Protection Agency. PESTICIDE FACT SHEET / Clofencet (1997); Bucholtz, D.L., (1988): Effect of environment and formulation on the absorption and translocation of fenridazon in wheat (Triticum aestivum L.). Plant Growth Regulation, 7: 65-73. Among substituted cinnolines the chemicals SC- 1271 (l-(4-chlorophenyl)-4-oxo-5- propoxycinnoline-3-carboxylic acid) and SC-2053 (l-(4-chlorophenyl)-4-oxo-5- (methoxyethoxy) cinnoline-3-carboxylic acid) developed by Orsan/DuPont stand out. SC-2053 represents the active substance sintofen used as a commercial chemical hybridizing agent CROISOR ^® 100. A CROISOR ^® 100 agent is applied when the length of a spike on the main stem is 14 mm to 18 mm long, with the dose of 13 1/ha to 15 1/ha (1.3 kg to 1.5 kg of active substance per hectare). The pollen grains of treated plants have a wavy surface and degenerated cytoplasm (general retardation) (Wong et al., 1995).

Use of derivatives of oxanilic acid intended for chemical hybridization is disclosed in Chakraborty, ., Devakumar, C. (2006): Ethyloxanilates as specific male gametocides for wheat (Triticum aestivum L.). Plant Breeding, 125: 441-447, wherein oxanilate ethyl esters were used as chemical hybridizing agents in the form of emulsions with an organic solvent (e. g. ethylene dichloride, methylnaphthalene, glycol ethers, cyclohexanone, anisole). Some derivatives of oxanilic acid and their use in the method for the induction of male sterility are disclosed also in GB patent No. 1 572 527 (Batch et al.). It is described therein that water dispersions or emulsions can be prepared by dissolving an active ingredient in an organic solvent. This document does not discuss a preparation of water-soluble preparations containing easily soluble or water-soluble derivatives of oxanilic acid and/or the water-soluble salts thereof.

Modern chemical hybridizing agents are further developed, especially in direction of achieving better selectivity of gametocidic activity and reduced influence on the environment. The purpose of the invention was to develop new environmentally friendly agents with gametocidic activity or chemical hybridizing agents and to reduce the introduction of volatile organic compounds into the environment and plants. Oxanilic acid was found to be an interesting basis for the preparation of agents for chemical hybridization of the invention, as their synthesis is simple and economically favourable and the acid function moreover offers a possibility of a further functionalisation with the purpose of producing water-soluble active substances. The present invention thus refers to a change in the structure of active substances in terms of their getting more easily-soluble and water-soluble, and to a preparation of water- soluble preparations that confer a more simple use for the application in the field in the form of water solutions.

Solution to the Technical Problem with Examples

The present invention refers to a chemical way of exploiting a heterosis in commercially significant hermaphrodite plant species, preferably from the family of grasses (lat. Poaceae), especially common wheat (lat. Triticum aestivum L.), in which easily soluble derivatives of oxanilic acid of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof are used as an active chemical hybridization substance.

The invention further refers to a method for the production of hybrid seeds of F] generation of commercially significant hermaphrodite plant species, preferably hermaphrodite cereals from the family of grasses, more preferably common wheat, with chemical hybridization comprising:

selection of seeds of both parent components of a commercially significant hermaphrodite plant species so that sowing of seeds of both parent components is possible separately in the form of one or several separate strips or optionally in the form of a mixture of mutually mixed seeds of both parent components,

wherein in the case of sowing a mixture of seeds the female component of a hybrid variety (line AA) expresses a natural or genetically engineered resistance against a herbicide, preferably a non-selective herbicide, and the male component of a hybrid variety (line BB) (pollinator) does not express such resistance and wherein in the case of sowing the seeds of both parent components in the form of a mixture inflorescence of a pollinator (line BB) occurs when the end of the premeiotic stage occurs in the female component of the hybrid variety (line AA) due to flowering synchronisation of both parent components and due to prevention of induction of a high percentage of male sterility in the pollinator,

induction of male sterility in the plants of the female component of the hybrid variety (line AA), wherein easily soluble derivatives of oxanilic acid of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof are applied as an active substance for chemical hybridization onto plants from the end of the premeiotic stage of the plants of the female component on and/or when the latter are in the meiotic stage:

(I)

wherein:

X represents a halogen atom, such as fluorine, chlorine, bromine or iodine, or a -CN group or hydrogen or an alkyl or fluoroalkyl group, preferably a bromine atom or a -CN group;

Y represents a group selected from the group comprising -OH and/or -OM, wherein M represents a cation suitable for the formation of an agriculturally acceptable water-soluble salt, such as alkali metal cation, e. g. Na ⁺ or K ⁺ , or tetraalkylammonium ion, preferably Na ⁺ ;

and optionally

in the case of sowing seeds in the form of a mixture of both parent components, after the completed fertilisation of the plants of the female component of the hybrid variety (line AA), wherein the female component expresses natural or genetically engineered resistance against herbicide, preferably a non-selective herbicide, and the male component (line BB) does not express such resistance, removing of the plants of the male component from the crop by applying a herbicide, preferably a non-selective herbicide, against which the plants of the female component express resistance, whereas the plants of the male component or pollinator do not have this resistance, onto plants and their locus.

The method of the invention can be used on commercially significant hermaphrodite plant species, preferably hermaphrodite cereals from the family of grasses (lat. Poaceae), especially common wheat (lat. Triticum aestivum L).

The expression "both parent components" as used herein denotes the seeds of the female component (line AA) and the male component (line BB) of commercially significant hermaphrodite plant species, especially hermaphrodite cereals from the family of grasses, preferably common wheat. According to one aspect of the invention, they were selected in a way that the female and male components have a suitable delay in development, so that inflorescence of the male component (pollinator) becomes noticeable at the end of the premiotic phase or the beginning of the meiotic stage of organogenesis of the female component (synchronisation of flowering). It is herewith enabled that the active chemical hybridization substance, i. e. the compound of general Formula (I) and/or its agriculturally acceptable water- soluble salt, is applied onto plants of the female component in the period from the completion of the premeiotic phase of the plants of the female component and/or when the latter are in the meiotic stage of organogenesis.

In a preferred embodiment, the method of the invention is carried out on common wheat (lat. Triticum aestivum L.). In the application phases of the active substance, i. e. the compound of general Formula (I) and/or the water-soluble agriculturally acceptable salts thereof, onto the plants, the spikes on the main stem of the female component (line AA) are 15-20 mm long, whereas the spikes on the main stem of the male component (line BB) are≤ 5 mm long. As female components (line AA), genotypes (cultivars) of the common wheat selected from the group comprising Ficko, Guarni, Sana, Inoui, Marija, Bologna are especially applicable for the use of the invention.

As the male component the cultivar Ludwig of the common wheat (lat. Triticum aestivum L.) is suitable within the meaning of the invention.

In order to exploit heterosis or production of hybrid seeds of commercially significant hermaphrodite plant species, preferably from the family of grasses (lat. Poaceae), especially preferably common wheat (lat. Triticum aestvum L.) by applying a chemical hybridizing agent, wherein the seeds of both parent components are sown in the form of a mixture, such female component needs to be selected, under the consideration of already mentioned synchronisation of flowering of plants of both parent components, that has a determinant (i. e. gene) for resistance against a herbicide, preferably a non-selective herbicide. In order to introduce resistance against N-phosphinomethy giycin (glyphosate) which is the most often used active substance in non-selective herbicides, there are various transgenic approaches known to a person skilled in the art (e. g. gene cp4-epsps, bar, gox); natural resistance (an endogenous gene) of plants is also used and it can be successfully induced by regenerating the plants on culture mediums with added said active substance - non-selective herbicide. Wheat resistant against non-selective herbicides and a method for the production thereof are disclosed in patent US 7,087,809 B2 (William, H. D. (2006) Natural herbicide resistance in wheat) and US patent application US 2009/0320151 (William, 2006, Kimberlee et al., 2009, Glyphosate-tolerant wheat genotypes).

A further aspect of the present invention is a method for producing a hybrid seed of F] generation of commercially significant hermaphrodite plant species, preferably common wheat, with chemical hybridization with an active substance of general Formula (I) and/or the agriculturally acceptable water-soluble salts thereof of the invention, wherein in the case of sowing seeds in the form of a mixture of both parent components, not only synchronisation of flowering of both parent components in order to prevent induction of a high percentage of male sterility in the pollinator in the phase of the chemical hybridization method is taken into consideration but moreover the seeds of both parent components are selected in a way that the female component expresses natural or genetically engineered resistance against a herbicide, preferably a non-selective herbicide, and the male component does not express such resistance. It is herewith achieved that after the fertilisation of the female component is completed the plants of the male component are eliminated by applying a herbicide, preferably a non-selective herbicide to obtain Fj seeds of the hybrid variety. A purer Fi generation of seeds can thus be obtained than could be if the well-known and prevailing method of sowing in strips known in the art were used, i. e. the Fj seed is obtained considerably without admixture of the BB line.

Suitable herbicides used for the removal of the male component after the fertilisation of the female component are sulphonylurea herbicides, such as rimsulforon and nicosulfuron, imidazolinone based herbicides, such as imazamox, and organophosphorous herbicides, such as gluphosinate or glyphosate. In a preferred embodiment the non-selective herbicide such as N-phosphonomethyl glycine (glyphosate) is used.

A mixture of mutually mixed seeds of both parent components can contain the seeds of female component mixed with the seeds of the male component in a ratio up to about 1 : 1. Applicable and feasible ratios are known to a person skilled in the art.

Recommended use in the production of hybrid seeds of common wheat is about 90 % of the seeds of the female component (line AA) and about 10 % of the seeds of the male component (line BB) based on the total mass of the seed mixture.

In producing hybrid barley by using cytoplasmic-genetic male sterility normally 95 % of line AA and 5 % of line BB are used. In one aspect or embodiment, the present invention is based on an interaction between the determinant that determines resistance to a non-selective herbicide (i. e. it either has a natural resistance to a non-selective herbicide or a gene introduced by genetic engineering), e. g. to N-phosphonomethyl glycine, and a chemical hybridizing agent based on easily soluble or water-soluble derivatives of oxanilic acid of general Formula I and/or agriculturally acceptable water-soluble salts thereof, which are a further aspect of the invention.

A further purpose of the invention is also to prepare chemical hybridizing agents on the basis of easily soluble derivatives of oxanilic acid that would be simple to synthesise and apply in the field. This was achieved by a development of easily soluble solutions of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof. By changing the structure of the active ingredient in terms of its increased water solubility, it is done away with a use of volatile organic solutions, unnecessary burdening of plants and environment with volatile organic substances in their application. The synthesis of compounds of the invention is also simple and economically acceptable. As the compounds of general Formula (I) and/or the salts thereof are easily soluble or water-soluble, water-soluble preparations can be prepared that have good applicability and allow a well-homogenised application onto plants. In order to increase absorption of the active ingredient from the water solution into a plant a surfactant can be added.

Easily soluble or water-soluble derivatives of oxanilic acid of the invention have a general Formula (I):

(I) wherein

X represents a halogen atom, such as fluorine, chlorine, bromine or iodine, or a -CN group or hydrogen or an alkyl or fluoroalkyl group, preferably a bromine atom or an iodine atom or a -CN group, more preferably a bromine atom or a -CN group;

Y represents a group selected from the group comprising -OH and/or -OM,

wherein M represents a cation suitable for the formation of an agriculturally acceptable water-soluble salt, such as alkali metal cation, e. g. Na ⁺ or K ⁺ , or tetra- alkylammonium ion, preferably Na ⁺ .

With the purpose of preparing easily soluble or water-soluble compounds of general Formula (I) and the salts thereof and of checking their phytotoxicity for the use as an agent for chemical hybridization of common wheat, the following easily soluble derivatives of oxanilic acid of general Formula (I) and the water-soluble salts thereof were prepared:

(I)

wherein:

Compound No. X Y

CHA1 4-Br -OEt ethyl 4-bromooxanilate

CHA2 4-CN -OEt ethyl 4-cianooxanilate

CHA3 4-F -OEt ethyl 4-fluorooxanilate

CHA4 4-Br -OH 4-bromooxanilic acid

CHA5 4-CN -OH 4-cianooxanilic acid

CHA6 4-F -OH 4-fluorooxanilic acid

CHA7 4-Br -O ^' (CH ₃ ) ₄ N ⁺ tetramethylammonium salt of 4- bromooxanilic acid CHA8 4-CN -O ^" (CH ₃ ) ₄ N ⁺ tetramethylammonium salt of 4- cianooxanilic acid

CHA9 4-F -0 ^" (CH ₃ ) ₄ N ⁺ tetramethylarnmonium salt of 4- fluorooxanilic acid

Results of tests of these compounds are presented in given examples.

A compound of general Formula (I), wherein X represents Br and Y represents an -OH group, i. e. 4-bromooxanilic acid, has been identified as particularly suitable compound for use of the invention.

The easily soluble or water-soluble derivatives of oxanilic acid of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof can be applied only on the plants of the female component, if the seeds of the female component were sown separately from the male component, e. g. as one or several strips of seed/plants of the female component separated from one or several strips of the seeds/plants of the male component, or on the plants that grew from a crop of a mixture of mutually mixed seeds of both parent components .

The method of the invention further refers to a method of producing hybrid seeds of Fi generation of commercially significant hermaphrodite plant species, especially common wheat, with chemical hybridization, wherein the seeds of a female component are sown in strips as known in the art. In this case, the step of inducing male sterility in the female component (line AA) is not followed by removal of the male component (line BB) from the seed crop.

The compounds of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof induce male sterility in hermaphrodite plant species particularly from the family of grasses, especially common wheat, from the end of their premeiotic stages onwards and/or in their meiotic stage in the application ranging from 0.1 kg/ha to 10 kg/ha and in a concentration from 0.01 % to 1.5 %.

The recommended dosage of the active substance for common wheat is from 1.4 kg/hectare to 3.5 kg/hectare or until toxic concentration occurs. It should be noted that water consumption per hectare should not exceed 700 1.

The compounds of general Formula (I) and/or agriculturally acceptable water-soluble salts thereof can be prepared in forms of various water-soluble preparations, e. g. water-soluble powders, water-soluble granules, water-soluble crystals or water- soluble concentrates, preferably in the form of water-soluble powders or water-soluble granules.

The preparation can also be in the form of a suspension concentrate, for instance in the case of reduced water consumption in the production of preparations and thus higher concentration of the active substance.

Water-soluble preparations can comprise one or more compounds of Formula (I) and/or agriculturally acceptable water-soluble salts thereof as the active ingredient. The preparations preferably comprise as an active ingredient one or more compounds selected from the group consisting of: 4-bromooxanilic acid, 4-cianooxanilic acid, tetramethylammonium salt of 4-bromooxanilic acid, tetramethylammonium salt of 4- cianooxanilic acid and tetramethylammonium salt of 4-fluorooxanilic acid and optionally further additives known in the art, such as surfactants, e. g. non-ionic surfactants and/or defoamers.

Application of easily soluble compounds of general Formula (I) and/or water-soluble salts thereof may require an addition of a surfactant (non-ionic surfactant) which enhances absorption of the active substance through leaf epidermis and addition of a silicon-based defoamer. The latter can be a constituent part of the preparation or can be added when preparing a water solution for application.

Suitable surfactants are known in the art, for instance the preparation Spartan ^® (produced by Interagro (UK) Ltd) or Genapol ^® UD 50 (produced by Clariant).

Suitable silicon-based defoamers are known to a person skilled in the art, the agent Abate (produced by Interagro (UK) Ltd) can be used for instance.

The conditions suitable for applying preparations based on the active substances of the invention are at temperatures above 5 °C, preferably at temperatures above 10 °C and relative humidity above 30 %, preferably above 50 %. Suitable and optimal conditions are known to a person skilled in the art.

The method of the invention allows for a simpler composition of a seed crop in the embodiment when sowing a mixture of mutually mixed seeds of both parent components (line AA + line BB): sowing in strips is no longer necessary, the space is better used, less time is spent for sowing, better pollination is achieved and more seeds of the desired F _! generation are produced with respect to the sown quantity of the mixture of mutually mixed seeds of both parent components. Moreover, treatment of plants of the female component with the chemical hybridizing agent is simpler, whereas special attention is needed when applying the agent in a crop sown in strips. In comparison with sowing in strips, more seeds of the desired F) generation are obtained as there are no strips of the so-called male component (pollinator), the seeds of which can be considered to be a side product in terms of production of hybrid seeds of F) generation.

Examples given in the continuation are for illustration purposes of the present invention and do not limit the scope of this invention in any way whatsoever. Unless otherwise specified, all percentages indicated in the patent application refer to mass percents.

Examples

Example 1: Synthesis of 4-bromooxanilic acid (CHA4) and preparation of an aqueous solution for application

4-bromoaniline (67.73 g; 0.394 mol) and diethyl oxalate (287.7 g; 1.969 mol) were weighted and added into a one-litre round-bottom flask and stirred for 24 hours at 1 10 °C. When the reaction was completed, the residual diethyl oxalate was partially evaporated under reduced pressure (60 mmHg). A product precipitated as a solid compound which was filtered off and washed on a suction filter with hexane. Pure ethyl 4-bromooxanilate was obtained in the form of a white powder (99.6 g, 93 mol%; ^l H NMR 6(ppm) 1.31 (t, 3H), 4.30 (q, 2H), 7.56 (d, 2H), 7.73 (d, 2H), 10.91 (s, 1H)).

The product was wetted with 35 mL of acetonitrile, 100 mL of water was added and 10 % water solution of NaOH was slowly added (500 mL in one hour) at a temperature of 0 °C and stirred at 20 °C for further 5 hours. The reaction mixture was neutralised with a cold 10 % aqueous solution of HC1 and the solid product was filtered off, rinsed on a suction filter with water and dried. 87.6 g (98 mol%) of the product, oxanilic acid derivative (I, X = Br, Y = -OH), 4-bromooxanilic acid (I) was obtained. 1H NMR 6(ppm) 7.53 (d, 2H), 7.75 (d, 2H), 10.84 (s, 1H)).

The aqueous solution for spraying was prepared by mixing 3 g of the product (I) (4- bromooxanilic acid) with 12 g of Spartan (Interagro (UK) Ltd) (agent for wetting a leaf surface). The mixture so obtained was dissolved in a corresponding quantity of water relative to the concentration and application surface (e. g. in 3 litres of water) and applied directly onto the plants in the field and their locus. Example 2: Preparation of water-soluble preparations - preparation of tetramethylammonium salt of 4-bromooxanilic acid of general Formula I

For the preparation of 3g of tetramethylammonium salt of 4-bromooxanilic acid 4- bromooxanilic acid (2.308 g) prepared by the method from Example 1 and tetramethylammonium hydroxide pentahydrate (1.713 g) were mixed in 200 mL of water and dissolved to the corresponding volume of the aqueous solution relative to the concentration and application surface (e. g. in 3 litres of water) and directly applied onto the plants in the field and their locus.

From the prepared compounds which were described above the following preparationswere prepared for spraying application:

Compound No. Preparation

CHA1 emulsion prepared from 3 g of CHA1/ 7.5 g of Genapol ^® UD 50

/24 mL of dimethylsulfoxide

CHA2 emulsion prepared from 3 g of CHA2/ 7.5 g of Genapol ^® UD-50

/120 mL of dimethylsulfoxide

CHA3 emulsion prepared from 3 g of CHA3/ 7.5 g of Genapol ^® UD- 50/135 mL of dimethylsulfoxide

CHA4 solution, 3 g of CHA4 in 12 g of Spartan ^® is dissolved in 3 litres of water

CHA5 solution, 3 g of CHA5 in 12 g of Spartan ^® is dissolved in 3 litres of water

CHA6 solution, 3 g of CHA6 in 12 g of Spartan ^® is dissolved in 3 litres of water

CHA7 solution, 3 g of CHA7 in 200 mL of water, 2.144 g of acid CHA3 and 2.120 g of Me ₄ NOH x 5 H ₂ O, pH - 9 CHA8 solution, 3 g of CHA8 in 200 mL of water, 2.308 g of acid CHA4 and 1.713 g of Me ₄ NOH x 5 H ₂ O, pH = 9

CHA9 solution, 3 g of CHA9 in 200 mL of water, 2.167 g of acid CHA5 and 2.064 g of Me ₄ NOH x 5 H ₂ O, pH = 9

CHA10- Sintofen CROISOR ^® 100 - following the manufacturer's control instructions

The preparations were used or applied as specified in Example 3.

Example 3: Efficiency of oxanilic acid derivatives of the invention compared to the standard chemical hybridizing agent - CROISOR ^® 100

The efficiency of oxanilic acid derivatives of general Formula (I) and/or the salts thereof in terms of reaching a high percentage of male sterility in common wheat (lat. Triticum aestivum L.) and their influence on the plant's height were tested in field conditions in comparison with the standard chemical hybridization agent CROISOR® 100 (active substance Sintofen, supplied by Saaten-Union Recherche S. A. R. L.). Tested factors were: location (L) (central region, north-eastern region of Slovenia), active substance (AS): ethyl 4-bromooxanilate (CHA1), ethyl 4-cianooxanilate (CHA2), ethyl 4-fluorooxanilate (CHA3), 4-bromooxanilic acid (CHA4), 4- cianooxanilic acid (CHA5), 4-fluorooxani ic acid (CHA6), tetramethylammonium salt of 4-bromooxanilic acid (CHA7), tetramethylammonium salt of 4-cianooxanilic acid (CHA8), tetramethylammonium salt of 4-fluorooxanilic acid (CHA9) and as control Sintofen (CROISOR ^® 100) (CHA10), dose (D) (Dl - 700 g/ha, D2 - 1400 g/ha, D3 - 2100 g/ha, D4 - 2800 g/ha, D5 - 3500 g/ha and control - 0 g/ha) and phenophase (FP) which was determined on the basis of the length of the spike (FP1 - 5 mm, FP2 - 10 mm, FP3 - 15 mm). Sowing was carried out on both locations in the first decade of October (genotype cv. Guarni - Florimond Desprez), crop's density amounted to 450 grains/m ² , the distance between rows was 12.5 cm. The trial consisted of eleven 30- metre long and 1 -metre wide strips. The first strip was for control purposes, whereas the remaining ten strips were intended for fifteen treatments. During vegetation, the crop was supplied with nitrogen in the form of 27 % calcium ammonium nitrate (EC 13/25, 70 - 90 kg N/ ha; EC 31/32, 50 - 70 kg N/ha; EC 47/49, 70 - 90 kg N/ha), the plants were protected with a sulfonylurea herbicide (EC 13/37), strobilurin and triazole based fungicides (EC 29/37 - strobilurin; EC 51/69 - triazole) and deltametrin based insecticide. The oxanilic acid derivatives and the standard chemical hybridizing agent were applied in a 0.1 % concentration. Application was performed in the morning, when relative air humidity was at least 50 % and air temperature exceeded 10 °C. The nozzles Lechler 1 10 - 04 positioned 30 cm above the plant habitus were used.

Male sterility was calculated by the formula (Sc - Sf) / Sc x 100, wherein Sc is the number of seeds per spike of an untreated plant and Sf is the number of seeds per spike of a treated plant. Apart from male sterility phytotoxicity was determined as well. The indicator of phytotoxicity was the plant height. Statistically significant differences and interactions were determined by the statistical software package STATGRAPHICS Centurion XVI.

The results have shown that the compound CHA4 - i. e. 4-bromooxanilic acid - induces the highest percentage of male sterility among the tested derivatives. The results are shown in Table 1. It is of utter importance that said derivative reaches better selectivity and lower toxicity in comparison with the standard chemical hybridizing agent. This means that the efficiency of the derivative CHA4 increases with the spike getting longer and that the plant height gets lower more slowly, which is shown in Figures 1 and 2.

The figures show the interaction AS x FP x D. Figure 1 shows selectivity of activity of the standard hybridizing agent and Figure 2 shows selectivity of activity of 4- bromooxanilic acid. Table 1 : Statistically significant differences and interactions

Plant height [cm] Male sterility [%]

Location (L) ** **

Active substance (AS) ** **

Phenophase (FP) ** **

Dose (D) ** **

Statistically significant All interactions except All interactions interactions LxD

Mean values of factors [cm, %]

Location (L)

North-eastern region 62.681 ± 0.1 106 ^b 70.8252 ± 0.3482 ³

Central region 66.2132 ± 0.1 106 ^a 58.2274 ± 0.3482 ^b

Active substance (AS)

CHA1 62.3586 ± 0.2473 ^g 76.7743 ± 0.7787 ^d

CHA2 68.4979 ± 0.2473 ^c 45.9719 ± 0.7787 ^fg

CHA3 63.9412 ± 0.2473 ^f 80.1045 ± 0.7787 ^c

CHA4 59.891 1 ± 0.2473 ^h 82.4494 ± 0.7787 ^ab

CHA5 70.5985 ± 0.2473 ^ab 50.7552 ± 0.7787 ^e

CHA6 64.7152 ± 0.2473 ^e 74.8315 ± 0.7787 ^d

CHA7 66.5369 ± 0.2473 ^d 47.3379 ± 0.7787 ^f

CHA8 71.0378 ± 0.2473 ^a 44.398 ± 0.7787 ^g

CHA9 70.3285 ± 0.2473 ^b 45.1039 ± 0.7787 ^g

CHAIO (Standard (Sintofen)) 36.5654 ± 0.2473' 97.5362 ± 0.7787 ³

Phenophase (FP)

FP1 65.6362 ± 0.1368 ^a 58.7951 ± 0.4309 ^c

FP2 64.3021 ± 0.1341 ^b 64.8542 ± 0.4223 ^b

FP3 63.403 ± 0.1353° 69.9296 ± 0.4262 ³

Dose (D)

Dl 68.3425 ± 0.1747 ^a 58.7505 ± 0.5503 ^e D2 65.3517 ± 0.1747 ^b 62.2927 ± 0.5503 ^d

D3 63.92 ± 0.1747 ^c 65.2263 ± 0.5503 ^c

D4 63.0617 ± 0.1747 ^d 67.2789 ± 0.5503 ^b

D5 61.5597 ± 0.1753 ^e 69.0831 ± 0.5521 ^{a ns} No statistically significant differences at P ≥0.05

* Statistical significance defined at P < 0.05 and **P <0.01

± Standard error

Example 4: Influence of genotype on the efficiency of 4-bromooxanilic acid (CHA4) and the standard chemical hybridizing agent

The influence of the genotype on the chemical induction of male sterility was determined for the oxanilic acid derivative which reached a statistically significant highest percentage of male sterility on both locations in the season 2009/10 (compound CHA4, 4-bromooxanilic acid) and for the standard chemical hybridizing agent (CHA) - Sintofen. In the season 2010/1 1 a field experiment with six various genotypes was carried out for this purpose in the central region. The genotypes were: cv. Ficko, Guarni, Sana, Inoui, Marija, Bologna, wherein each genotype (inbred line) was sown in the shape of five consecutive plots with eight rows of 7.5 m (distance between the rows was kept at 12.5 cm). The fifth plot in each cultivar was intended for control. Both active substances (sintofen and the compound CHA4) were applied in the recommended dose and under recommended conditions for the standard hybridizing agent (1400 g/ha) in the form of 0.1 % suspension. Apart from the genotype and the active substance the influence of the phenophase on the induction of male sterility and plant's height was also studied. First application was carried out when the length of the spike on the main stem reached 5 - 10 mm and the next application was carried out when said length reached to 15 - 20 mm. Agrotechnical measures, application technique of the active substance, the method of determining the percentage of male sterility and plant's height did not differ from those in the field experiment for testing the efficiency of easily soluble oxanilic acid derivatives of the invention compared to the standard chemical hybridizing agent. The results of the experiment are gathered in Table 2.

Table 2: Influence of genotype on the efficiency of 4-bromooxanilic acid (CHA4) and the standard chemical hybridizing agent

MS - Male steri ity expressed in percents, V - plant's height expressed in centimetres

Example 5: Influence of 4-bromooxanilic acid (CHA4) and the standard chemical hybridizing agent on the synchronisation of flowering

In the field experiment carried out in order to determine the influence of a genotype on the efficiency of the CHA4 derivative and the standard chemical hybridizing agent the pollinator profile needed for the production of hybrid seeds of common winter wheat with the use of oxanilic acid derivatives was determined. Based on the date of flowering in the season 2009/10 six female components (cv. Ficko, Guarni, Sana, Inoui, Marija, Bologna) were selected out of 60 genotypes and one pollinator (cv. Ludwig). The strips of female components (9 AA) and the pollinator (<$ BB) were sown in an alternate manner, so that five consecutive plots of each female component (8 x 12,5 cm x 750 cm) were enclosed from each side by five consecutive plots of the pollinator. Flowering synchronisation was determined by the share of spikes of the female component that culminated (lemna and palea open at an angle exceeding 30 degrees) in the time when at least a half of the pollinator's spikes extrude anthers. In the case when less than 50 % of the spikes of the line AA culminate in the time when less than one half of the spikes of the line BB (visible stamina) flower, we cannot talk about a successful flowering synchronisation of both parent components of a potential hybrid variety. The results of the experiment are gathered in Table 3.

Table 3: Influence of 4-bromooxanilic acid (CHA4) and a standard chemical hybridizing agent on synchronisation of flowering

CHA4 Standard - CROISOR ^® 100

Phenophase 1 Phenophase 2 Phenophase 1 Phenophase 2

Genotype

Culmination Culmination Culmination Culmination

[%] l%] [%] [%]

Ficko 58 53 19 47

Guarni 57 65 27 60

Sana 47 55 44 50

Inoui 43 70 29 61

Marija 48 55 22 39

Bologna 64 51 17 41