WO2000063356A2

WO2000063356A2 - Herbicidal seed treatment

Info

Publication number: WO2000063356A2
Application number: PCT/EP2000/003451
Authority: WO
Inventors: Carroll Moseley; Rakesh Jain
Original assignee: Novartis Erfindungen Verwaltungs GmbH; Novartis AG; Syngenta Participations AG
Current assignee: Novartis Pharma GmbH Austria; Novartis AG; Syngenta Participations AG
Priority date: 1999-04-19
Filing date: 2000-04-17
Publication date: 2000-10-26
Anticipated expiration: 2001-10-19
Also published as: AR023523A1; AU5392000A; WO2000063356A3

Abstract

The present invention relates to the application of a herbicide as a seed treatment comprising the herbicide, preferably to select herbicide tolerant plants. The invention further relates to seed treatment compositions comprising such a herbicide, to methods of treating seeds with such a seed treatment composition, to seeds treated with a seed treatment composition of the present invention and to methods of using such seed treatments.

Description

HERBICIDAL SEED TREATMENT

The present invention relates to the field of seed treatment, in particular to herbicidal compounds applied as seed treatment. For example, such seed treatments are used to select seeds tolerant to the herbicide.

The size of the acreage planted with transgenic crops has greatly expanded in the last few years and transgenes are being bred into an increasingly large number of elite lines in different crops. This requires efficient ways to follow the transgene, for example during the various steps leading to a commercial variety, such as breeding processes, or to assure the purity of transgenic seeds during inbred or hybrid seeds production. This can be done using molecular analysis methods, such as Southern blot analysis or PCR amplification, or immunological detection methods. However, such methods require harvesting plant material, bringing it to a laboratory and carrying out the analysis, which is time consuming, expensive and subject to errors, such as detecting false positives or missing the detection of false negatives.

A large proportion of transgenic plants comprise a trait conferring upon the plant tolerance to a herbicide, alone or in combination with another transgenic trait. The herbicide tolerance trait often co-segregates with the other transgenic trait and can be used to select plants comprising the transgene in the field and to eliminate non-transgenic plants without relying upon molecular analysis. However, this usually requires planting seeds on large acreages and growing and spraying plants with high amounts of the herbicide in order to be able to efficiently select plants tolerant to the herbicide. This is also expensive, time consuming and often environmentally unfriendly.

An elegant way to circumvent these difficulties would be to apply the herbicide directly to the seeds prior to planting and germination. However, generally, herbicides are not effective as seed treatments. Thus, although a lot of efforts have been directed to the development of herbicides applied as seed treatments, this form of application has remained unsuccessful.

There is therefore a long felt and unfulfilled need in the agricultural field for reliable, cheap, effective and environmentally friendly ways to apply herbicides, in particular to select seeds tolerant to a herbicide. The present invention addresses the need for novel ways to apply herbicides, in particular to efficiently select plants tolerant to a herbicide. A feature of the invention is the use of a seed treatment comprising a PPO herbicide. To date most herbicides are not appropriate for selecting seeds tolerant to the herbicide when applied as seed treatments. It is therefore an unexpected and surprising discovery of the inventors of the present invention that PPO herbicides are well-suited to select tolerant seeds when incorporated in a seed treatment composition and applied to seeds prior to planting.

An advantage of the invention is that the early growth of herbicide sensitive seeds is inhibited, whereas herbicide tolerant seeds grow unhindered. A further advantage of the present invention is that, by following the teachings of the present invention, there is no need to carry out molecular or immunologicai analysis of plant material or to spray plants with the herbicide. The present invention therefore discloses an inexpensive, easy to implement, quick and reliable way to select herbicide tolerant seeds and plants.. The burden on the environment is also reduced due to the low rates of active ingredient required in an application as seed treatment.

The present invention is particularly useful in the various steps leading to commercial seed production, such as for example backcrossing the PPO tolerance trait into elite lines or for inbred or hybrid seed production. The present invention is also useful for the identification of homozygous progeny of a transformed line or for seed amplification of a transformed homozygous line.

The present invention therefore encompasses seed treatment compositions comprising a PPO herbicide, methods of treating seeds with compositions of the present invention, seeds treated with such seed treatment compositions and methods of using such seeds.

The present invention thus provides:

A method for selecting plants tolerant to a PPO herbicide comprising applying to a population of seeds a seed treatment comprising an amount of a PPO herbicide effective to inhibit the early growth of a seed sensitive to said PPO herbicide, placing said population of seeds under conditions sufficient for germination, and selecting plants that grow. In a preferred embodiment, the population of seeds is obtained from a cross, wherein one of the parents in the cross is a PPO herbicide tolerant plant. In another preferred embodiment, the population of seeds is obtained from the self-pollination of a PPO herbicide tolerant plant. In another preferred embodiment, seed tolerant to a PPO herbicide comprises a heterologous polynucleotide encoding a PPO, preferably under the control of a promoter capable of directing the expression of said heterologous polynucleotide in said seed tolerant to a PPO herbicide or in emerging seedlings derived from said seed tolerant to a PPO herbicide. Preferably, the heterologous polynucleotide encodes a PPO herbicide tolerant PPO, preferably derived from a bacterium. In yet another preferred embodiment, the heterologous polynucleotide encodes a modified PPO, preferably derived from a plant, preferably Arabidopsis.

In another preferred embodiment, the population of seeds is derived from a monocotyledonous plant, preferably selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice and barley. In another preferred embodiment, the population of seeds is derived from a dicotyledonous plant, preferably selected from the group consisting of soybean, cotton, tobacco, sugar beet and oilseed rape. In another preferred embodiment, the step of applying to said population of seeds a seed treatment comprises soaking said population of seeds in a composition comprising a PPO herbicide or coating seeds of said population with a composition comprising a PPO herbicide.

In another preferred embodiment, a PPO herbicide is selected from the group consisting of an aryluracil, a diphenylether, an oxadiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole and an other heterocyclyl pyrazole derivative, a pyridyl amide derivative, a 3-substituted-2- aryl-4,5,6,7-tetrahydroindazole, a phenopylate and O-phenylpyrrolidino- and O- phenylpiperidinocarbamate analogs of said phenopylate. Preferably, a PPO herbicide is a N-phenyl-uracil, more preferably benzoic acid 2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4- (trifluoromethyl)-1(2H)-pyιimidinyl]- 1,1-dimethyl-2-oxo-2-(2-propenyloxy)ethyl ester (9CI, proposed common name: butafenacil). The present invention further provides:

A treated seed comprising an amount of a PPO herbicide effective to inhibit the early growth of a PPO herbicide sensitive seed. In a preferred embodiment, the seed is derived from a monocotyledonous plant, preferably selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice and barley. In another preferred embodiment, the seed is derived from a dicotyledonous plant, preferably selected from the group consisting of soybean, cotton, tobacco, sugar beet and oilseed rape.

In another preferred embodiment, a PPO herbicide is selected from the group consisting of an aryluracil, a diphenylether, an oxadiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole and an other heterocyclyl pyrazole derivative, a pyridyl amide derivative, a 3-substituted-2- aryl-4,5,6,7-tetrahydroindazole, a phenopylate and O-phenylpyrrolidino- and O- phenylpiperidinocarbamate analogs of said phenopylate. Preferably, a PPO herbicide is a

N-phenyl-uracil, more preferably benzoic acid 2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-

(trifluoromethyl)-1 (2H)-pyrimidinyl]- 1 ,1-dimethyl-2-oxo-2-(2-propenyloxy)ethyl ester (9CI, proposed common name: butafenacil).

The present invention further provides:

A composition comprising an amount of a PPO herbicide effective to inhibit the early growth of a PPO herbicide sensitive seed, wherein said composition is applied to a seed as a seed treatment.

In a preferred embodiment, a PPO herbicide is selected from the group consisting of an aryluracil, a diphenylether, an oxadiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole and an other heterocyclyl pyrazole derivative, a pyridyl amide derivative, a 3-substituted-2- aryl-4,5,6,7-tetrahydroindazole, a phenopylate and O-phenylpyrrolidino- and O- phenylpiperidinocarbamate analogs of said phenopylate. Preferably, a PPO herbicide is a

(trifluoromethyl)-l (2H)-pyrimidinyl]-1 ,1 -dimethyl-2-oxo-2-(2-propenyloxy)ethyl ester (9CI, proposed common name: butafenacil).

In another preferred embodiment, the amount of a PPO herbicide is between 1 and 160 g ai/ kg seed, preferably between 2 and 40 g ai/ kg seed, more preferably between 5 and 20 g ai/ kg seed.

In another preferred embodiment, a composition of the present invention further comprises additional ingredients, such as formulations (dry flowables (DF), liquid flowables (LF), true liquids (TL), emulsifiable concentrates (EC), dusts (D), wettable powders (W P) and the like), adhesives, carriers, surfactants, application-promoting adjuvants or other ingredients customarily employed in the art of formulation.

In another preferred embodiment, a composition of the present invention further comprises other herbicides or insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof.

The present invention further provides:

A method comprising applying to a seed a composition of the present invention.

The present invention further provides:

A method for producing PPO tolerant seeds comprising a) producing PPO tolerant plants by transforming plants with a heterologous polynucleotide encoding a PPO b) crossing two plants by pollinating a recipient plant with pollen from a donor plant, wherein at least one of the two plants is tolerant to PPO herbicide c) allowing the pollinated plant to set seeds d) harvesting said seeds; and, optionally e) testing for PPO tolerance by applying a seed treatment comprising a PPO herbicide to seeds resulting from each cross.

In a preferred embodiment, the donor plant and the recipient plant are identical, i.e. are from a self-pollinating species.

In another preferred embodiment, the donor plant and the recipient plant are from a cross- pollinating species.

In yet another preferred embodiment, the donor and/or the recipient plant are from an elite line or from a line for inbred or hybrid seed production.

DEFINITIONS

For clarity, certain terms used in the specification are defined and presented as follows:

"Early growth": means herein the developmental stages of plant growth, preferably from the planting of the seed through the germination of the seeds and up to the appearance of the cotyledons or first true leaves.

Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only.

"Expression cassette" as used herein means a DNA molecule designed so that a polynucleotide of interest inserted herein can be transcribed and, optionally translated, in an appropriate host cell. The expression cassette typically comprises regulatory elements, such as a promoter capable of directing expression of the polynucleotide operably linked to the polynucleotide. The polynucleotide optionally may be operably linked to 3' sequences, such as 3 untranslated regions (3'UTR), e.g. 3' regulatory sequences or termination signals, and 5' untranslated regions (5'UTR). The expression cassette also may comprise additional sequences required for proper translation of a coding sequence comprised in the polynucleotide, such as e.g. ribosome binding sites. The expression cassette comprising the polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell. The expression of the polynucleotide in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue or organ or stage of development. A nuclear expression cassette is usually inserted into the nuclear genome of a plant and is capable of directing the expression of a particular polynucleotide from the nuclear genome of said plant. A plastid expression cassette is usually inserted in to the plastid genome of a plant and is capable of directing the expression of a particular polynucleotide from the plastid genome of said plant. For example a promoter capable of directing the expression of a particular polynucleotide from the plastid genome of said plant is a promoter recognized by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, or is a transactivator- mediated promoter. In a preferred embodiment, a plastid transgene comprises one gene transcribed from a single promoter. In a preferred embodiment, two or more genes transcribed from a single promoter in an operon-like polycistronic gene. "Gene" refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

"Herbicide" is a chemical substance used to kill or inhibit the growth of plants, plant cells, plant seeds, or plant tissues. In the context of the present invention, a preferred herbicide is a chemical substance that inhibits the enzymatic activity of protoporphyrinogen IX oxidase (PPO) in a plant and is referred herein as a "PPO herbicide". "Heterologous" as used herein means "of different natural origin" or represents a non- natural state. For example, if a host cell is transformed with a polynucleotide derived from another organism, particularly from another species, that polynucleotide is heterologous with respect to that host cell and also with respect to descendants of the host cell which carry that gene. Similariy, heterologous refers to a polynucleotide derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. a different copy number, under the control of different regulatory elements or at a different genomic location. A transforming polynucleotide may comprise a heterologous coding sequence, or heterologous regulatory elements. Alternatively, the transforming polynucleotide may be completely heterologous or may comprise any possible combination of heterologous and endogenous polynucleotides.

"Operably linked" refers to two DNA sequences that are related physically or functionally, for example such that one of the DNA sequences affects expression of the other.

A "plant" refers to any plant or part of a plant at any stage of development, such as e.g. fruits, leaves, stems, seeds, or roots. Therein are also included cuttings, cell or tissue cultures. As used in conjunction with the present invention, the term "plant" includes, but is not limited to, whole plants, plant cells, plant organs, plant tissues, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units.

"PPO" protoporphyrinogen IX oxidase.

"PPO herbicide sensitive seed or plant" refers preferably to a non-transgenic seed or plant, preferably from the same species as a PPO herbicide tolerant seed or plant of the present invention.

Promoter DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.

"Regulatory elements" refer to DNA sequences involved in the expression of a polynucleotide. Regulatory elements comprise a promoter operably linked to the polynucleotide of interest, and may also include 5' and 3' untranslated regions (UTR) or termination signals. They also typically encompass sequences required for proper translation of the polynucleotide, such as, e.g., ribosome binding sites.

'Tolerance": the ability to continue normal growth or function when exposed to a herbicide in amounts that affect normal growth or function in sensitive plants. In the context of the present invention, "PPO herbicide tolerant seeds or plants" are generally transgenic.

"Transoenic": means plants containing a heterologous DNA or polynucleotide that preferably comprises a suitable promoter operatively linked to the DNA or polynucleotide of interest. A transgenic plant may for example comprise a heterologous polynucleotide that confers upon the plant tolerance to a PPO herbicide. The present invention discloses for the first time the use of a seed treatment comprising a PPO herbicide, preferably to select plants tolerant to the PPO herbicide. Herbicides are generally not widely used in seed treatments and, more particularly, herbicides are not used as seed treatment to select plants tolerant to the herbicide. It is therefore surprising and unexpected that PPO herbicides are effective at selecting tolerant plants when applied as a seed treatment as described in the present invention. The application of a PPO herbicide as a seed treatment according to the present invention provides a simple, inexpensive and reliable way to select plants tolerant to PPO herbicides. The present invention therefore offers a significant improvement over existing methods to select herbicide tolerant plants which are based upon the use of costly and work intensive molecular or immunological analysis to select tolerant plants or which require spraying large acreages of plants with high amounts of herbicide.

Disclosed herein are seed treatment compositions comprising a PPO herbicide, methods of treating seeds with compositions of the present invention, seeds treated with a seed treatment compositions of the present invention and methods of using such seeds.

1. PPO herbicides

PPO herbicides affect plants by inhibiting the activity the enzyme protoporphyrinogen IX oxidase. This leads to the accumulation of protoporphyrinogen IX in the chloroplast. This accumulation is thought to lead to leakage of protoporphyrinogen IX into the cytosol where it is oxidized by a peroxidase activity to protoporphyrin IX. When exposed to light, protoporphyπ^'n IX can cause formation of singlet oxygen in the cytosol. This singlet oxygen can in turn lead to the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al., Plant Physiol. 702: 881 (1993)).

The inventors postulate that conventional herbicides, particularly those herbicides that inhibit amino acids biosynthesis, such as sulfonylureas, glufosinate and glyphosate, are not appropriate as seeds treatments to select tolerant plants because of their mode of action. The inventors further postulate that sufficient reserves of amino acids are present in seed storage compartments which could allow seeds to germinate and survive the effect of a herbicide present only in a seed treatment. In contrast, PPO herbicides are shown in the present invention to have full efficacy to inhibit the early growth of sensitive seeds without significantly affecting the growth of tolerant seeds. The inventors of the present invention speculate that this surprising and unexpected successful use of PPO herbicides as seed treatment to select PPO tolerant plants is due to their different mode of action. Accordingly, any PPO herbicide is appropriate as a seed treatment according to the present invention. PPO herbicides include many different structural classes of molecules (Duke et al., Weed Sci. 39: 465 (1991); Nandihalli etal., Pesticide Biochem. Physiol. 43: 193 (1992); Matringe etal., FEBS Lett. 245: 35 (1989); Yanase and Andoh, Pesticide Biochem. Physiol. 35: 70 (1989)), including the diphenylethers {e.g. acifluorfen 5-[2-chloro-4- (trifluoromethyl)phenoxy]-2-nitrobenzoic acid; its methyl ester; or oxyfluorf en 2-chloro-1-(3- ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}; oxadiazoles (e.g. oxadiazon 3-[2,4-dichloro-5- (1 -methylethoxy)phenyl]-5-(1 ,1-dimethylethyl)-1 ,3,4-oxadiazol-2-(3/-/)-one); cyclic imides (e.g. S-23142 Λ-(4-chloro-2-f luoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim (MK 616) Λ/-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide); phenyl pyrazoles (e.g. TNPP-ethyl ethyl 2-[1 -(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5- oxyjpropionate; M&B 39279 1 -(2,6-dichloro-4-trifluoromethylphenyl)-4-cyano-5- aminopyrazole); pyridyl amide derivatives (e.g. LS 82-556 as disclosed in EP-A-0 112 262); and phenopylate and its O-phenylpyrrolidino- and O-phenylpiperidinocarbamate analogs and bicyclic triazolones as disclosed in the international patent application WO 92/04827; EP-A-0532 146). The diphenylethers of particular significance are those having the general formula

C -d- -S- ^(Formuιaι)' wherein R equals -COONa (Formula II, acifluorfen-sodium), -CONHSO₂CH₃ (Formula III fomesafen) or -COOCH₂COOC₂H₅ (Formula IV, fluoroglycof en-ethyl; see Maigrot etal., Brighton Crop Protection Conference-Weeds: 47-51 (1989)). Additional diphenylethers of interest are those where R equals:

NOCH₂COOCH₃

CCH₂OCH₃

/

(Formula IVa; see Hayashi etal., Brighton Crop Protection Conference-Weeds: 53-58

(1989)).

An additional diphenylether of interest is one having the formula:

(Formula IVb; bifenox, see Dest etal., Proc. Northeast Weed Sci. Conf. 27: 31 (1973)). A further diphenylether of interest is one having the formula:

(Formula IVc; oxyfluorfen; see Yih and Swithenbank, J. Agric. Food Chem., 23: 592 (1975)).

Yet another diphenylether of interest is one having the formula:

CH₃

(Formula IVd; lactofen, see page 623 of 'The Pesticide Manual", 10^th ed., ed. by C.D.S.

Tomlin, British Crop Protection Council, Surrey (1994)).

Also of significance are the class of herbicides known as imides, having the general formula:

(Formula V),

wherein Q equals 04- (Formula VI), or

(Formula VII), or

(Formula VIII), or

(Formula IX), or

ormula IXa), or

(Formula IXb)

(see Hemper et al. (1995) in "Proceedings of the Eighth International Congress of Pesticide Chemistry", Ragdale etal., eds., Amer. Chem. Soc, Washington, D.C., pp. 42-48 (1994)); and R equals H, Cl or F, R₂ equals Cl and R₃ is an optionally substituted ether, thioether, ester, amino or alkyl group. Alternatively, R₂ and R₃ together may form a 5 or 6 membered heterocyclic ring. Examples of imide herbicides of particular interest are:

(Formula Vila; fluthiacet-methyl, see Miyazawa etal., Brighton Crop Protection Conference- Weeds, pp. 23-28 (1993)),

(Formula X; sulfentrazone, see Van Saun etal.,

Brighton Crop Protection Conference-Weeds, pp. 77-82 (1991)),

(Formula Xa, azafenidin, see 'The Pesticide Manual",

11th ed., ed. by C.D.S. Tomlin, Entry No. 37, 1997),

(Formula Xb, oxadiargyl, see 'The Pesticide

Manual", 11th ed., ed. by C.D.S. Tomlin, Entry No. 538, 1997), (Formula Xc, profluazole of DuPont, Reg. No.

[190314-43-3]),

(Formula XI, flupropacil),

(Formula XII, pyraflufen),

(see Miura et al., Brighton Crop Protection Conference-

Weeds: 35-40 (1993)),

(Formula XIV, flumiclorac-

,

pentyl), (Formula XV, flumipropyn), and

(Formula XVI, flumioxazin)

The herbicidal activity of the above compounds is described in the Proceedings of the 1991 Brighton Crop Protection Conference, Weeds (British Crop Protection Council) (Formulae X and XVI), Proceedings of the 1993 Brighton Crop Protection Conference, Weeds (British Crop Protection Council) (Formulae XII and Vila), U.S. Patent No. 4,746,352 (Formula XI) and Abstracts of the Weed Science Society of America vol. 33, pg. 9 (1993)(Formula XIV). The most preferred imide herbicides are those classified as aryluracils and having the general formula

(Formula XVII),

wherein R signifies the group (C₂.₆-alkenyloxy)carbonyl-Cι^-alkyl, as disclosed in U.S. Patent No.5,183,492, herein incorporated by reference. Also of significance are herbicides having the general formula: (Formula XVIII; thiadiazimin), (see Weiler etal.,

Brighton Crop Protection Conference - Weeds, pp. 29-34 (1993));

(Formula XIX; carfentrazone), (see

Van Saun etal., Brighton Crop Protection Conference-Weeds: pp. 19-22 (1993)); N-substituted pyrazoles of the general formula:

(Formula XX),

wherein Ri is CrC₄-alkyl, optionally substituted by one or more halogen atoms;

R₂ is hydrogen, or a d-C -alkoxy, each of which is optionally substituted by one or more halogen atoms, or

R_n and R₂ together form the group -(CH₂)„-X-, where X is bound at R₂;

R₃ is hydrogen or halogen,

R₄ is hydrogen or Cι-C₄-alkyl,

R₅ is hydrogen, nitro, cyano or the group -COOR₆ or -CONR₇R_β, and

R₆ is hydrogen, CrC₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl; and R₇ and R₈ independently of each other are hydrogen or Cι-C₆-alkyl or

R₇ and R₈ together with the nitrogen to which they are attached form a morpholino, piperidino or a pyrrolidino group (see international patent publications WO 94/08999, WO 93/10100, and U. S. Patent No. 5,405,829 assigned to Schering); N-phenylpyrazoles, such as:

(Formula XXI; nipyraclofen), (see page 621 of 'The Pesticide

Manual", 9^th ed., ed. by C.R. Worthing, British Crop Protection Council, Surrey (1991 )); and 3-substituted-2-aryl-4,5,6,7-tetrahydroindazoles (Lyga etal. Pesticide Sci. 4229-36 (1994)).

(Formula XXIa; BAY 11340).

Also of significance are phenylpyrazoles of the type described in WO 96/01254 and WO 97/00246, both of which are hereby incorporated by reference (Formula XXII). Also of significance are pyridyl pyrazoles such as the following: (Formula XXIIIa, described in WO 98/21199) and

(Formula XXIIIb, described in WO 98/42698).

In a preferred embodiment, a PPO herbicide is a N-Phenyl-uracil, preferably benzoic acid 2- chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1(2H)-pyrimidinyl]-1 ,1-dimethyl- 2-oxo-2-(2-propenyloxy)ethyl ester (9CI, proposed common name: butafenacil):

(Formula XXIV; proposed common name: butafenacil)

2. Seed treatment compositions

In a preferred embodiment, the present invention discloses seed treatment compositions comprising a PPO herbicide in an amount effective to inhibit early growth of a PPO sensitive seed or plant. This means that such sensitive seeds fail to germinate or that plants germinating from sensitive seeds are affected in their development by the PPO herbicide. For example, sensitive plants are stunted, fail to develop cotyledons, or develop cotyledons but fail to develop true leaves or fail to produce normal roots. Sensitive plants may in some cases grow true leaves but do not further develop. In contrast, PPO herbicide tolerant seeds or plants germinate, form fully developed cotyledons, set roots and further develop normally. It is possible, however, that tolerant seed or plants show slight damage, such as lesions or traces of bums, that usually disappear as the plant grows and develops. This may be due to varying growth conditions, such as soil composition, light or temperature. Preferred rates of applications for PPO herbicides as seed treatment are between 1 and 160 grams of active ingredient (a.i.) per 100 kg of seeds. More preferred rates are between 2 and 40 grams ai per 100 kg of seeds, yet more preferred rates are between 5 and 20 grams ai per 100 kg of seeds. The rate of PPO herbicide applied to seed may vary depending for example on the crop, the soil and the climatic conditions in which the seeds are to be planted. It is well within the skills of an artisan in seed treatment to adapt the amount of PPO herbicide to any particular situation to obtain satisfactory results without undue experimentation.

A seed treatment of the present invention may only comprise a PPO herbicide dissolved in a solvent, for example water. The seed treatment may also comprise two or more different PPO herbicides, for example dissolved in a solvent. A seed treatment of the present invention may also further comprise additional ingredients, such as formulations (dry flowables (DF), liquid flowables (LF), true liquids (TL), emulsifiable concentrates (EC), dusts (D), wettable powders (WP) and the like), adhesives, carriers, surfactants, application- promoting adjuvants or other ingredients customarily employed in the art of formulation. Also, protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation.

Seed treatments of the present invention may also further comprise other herbicides or may further comprise insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD^®), methalaxyl (Apron^®), and pirimiphos-methyl (Actellic^®). Overcoat product may also be further added to the seeds in particular for crops such as for example cotton and canola.

The present invention is exemplified with corn but one skilled in the art would be able to adapt the teachings of the present invention to prepare seed treatment comprising a PPO herbicide applicable to seeds of any other crop without undue experimentation. Such crops are for example, but are not limited to, agronomically important crops, i.e., angiosperms and gymnosperms such as Arabidopsis, sugar cane, soybean, barley, cotton, tobacco, sugar beet, oilseed rape, maize, wheat, sorghum, rye, oats, turf and forage grasses, millet, forage and rice and the like. Particularly preferred are agronomically important crops, i.e., angiosperms and gymnosperms such as soybean, cotton, sugar beet, oilseed rape, maize, wheat, barley, sorghum, and rice.

3. Methods of making treated seeds

In another preferred embodiment, the present invention discloses methods of treating seeds with a seed treatment composition of the present invention. Seed treatments are applied by different methods, for example by soaking the seeds in the appropriate composition, by coating the seeds with the composition or by dusting seeds in the composition. Seed treatments of the present invention are suitable for example for the following formulations: dry flowables (DF), liquid flowables (LF), true liquids (TL), emulsifiable concentrates (EC), dusts (D), wettable powders (WP) and others, which are applied to the seeds. For example, MaximXL (fludioxonil + mefenoxam) is applied as a flowable treatment typically on com seeds and ApronMaxx (fludioxonil + mefenoxam) is applied as an EC and is typically used on soybeans.

Commercial formulations are normally diluted with water to allow enough liquid to spread the herbicide over the seed coat. For example, com plants typically use a total volume of 8-12 fl oz / cwt of seed. For soybeans this would be about 6-8 oz. For example, seeds are treated in a Hege 11 lab treater. This unit spins the seed around an inner wall and then the treatment is atomized and sprayed by a spinning disc in the center. The typical treatment process is a two step procedure, 1. primary application, where the seed and treatment first meet, and 2. secondary application, where the seeds are mixed to improve coverage of the treatment liquid.

4. PPO tolerant seeds

In another preferred embodiment, the present invention further discloses seeds which are treated with a PPO herbicide applied as seed treatment. A population of seed treated according to the present invention may comprise PPO herbicide tolerant and PPO herbicide sensitive seeds. PPO herbicide tolerant plants are generally produced by genetic engineering methods and such plants have been extensively described in WO 95/34659 and WO 97/32011 , both incorporated herein by reference. Preferably, PPO herbicide tolerant seeds of the present invention comprise a heterologous polynucleotide encoding a PPO. In a preferred embodiment, such polynucleotide encodes a herbicide tolerant PPO, preferably derived from a bacterium or encodes a modified PPO, preferably derived from a plant. A heterologous polynucleotide is typically operably linked to a promoter capable of directing the expression of the polynucleotide. Such polynucleotide is preferably included in an expression cassette, which is preferably stably integrated in the nuclear or plastid genome of the seed or plant.

In contrast, PPO herbicide sensitive seeds or plants generally refer to seeds or plants which are not tolerant to a PPO herbicide, preferably because they do not comprise a heterologous polynucleotide encoding a PPO as described above. Such PPO herbicide sensitive seeds or plants may be non-transgenic, wild-type plants, or may comprise a transgene that does not confer upon the seed or plant tolerance to a PPO herbicide. In a preferred embodiment, seeds of the present invention further comprise a transgene conferring upon such seeds another trait which preferably co-segregates with the trait conferring tolerance to a PPO herbicide. Examples of such trait are tolerance to another herbicide, resistance for bacterial, fungal, or viral disease, nematode resistance, insect resistance, enhanced nutritional quality, such as oil, starch and protein content or quality, improved performance in an industrial process, altered reproductive capability, such as male sterility or male fertility, yield stability and yield enhancement. Other transgenes transferred are also for the production of commercially valuable enzymes or metabolites in plants.

Treated seeds described herein for planting purposes are preferably containerized, e.g., placed in a bag or other container for ease of handling and transport.

5. Methods of using treated seeds

In another preferred embodiment, the present invention discloses methods of using treated seeds of the present invention. The present invention is beneficially used in any event where PPO herbicide tolerant plants are selected from PPO herbicide sensitive plants and is for example used on seeds originating from any self-pollination or cross-pollination of a PPO herbicide tolerant plant. PPO herbicide sensitive seeds originated from the cross and not carrying the PPO herbicide tolerant trait or seeds contaminations accidentally present in a populations of seeds obtained from the cross are thus eliminated. The present invention is for example useful for the identification of homozygous progeny of a transformed line. In this case, a primary transformant (T1 plants) which is tolerant to a PPO herbicide is self-pollinated. The resulting seeds are treated according to the present invention and only plants heterozygous or homozygous for the PPO herbicide tolerance trait (T2 plants) germinate and grow normally. Such plants are further allowed to self-pollinate and the resulting seeds are again treated according to the present invention. The ratio tolerant sensitive plants is determined for plants derived from each T2 plant and homozygous plants are identified. Similarly, once a homozygous plant has been identified, such plant can be self-pollinated and the resulting seeds treated according to the present invention. Thus, only PPO herbicide tolerant plants germinate and grow, thereby allowing for the amplification of homozygous, homogenous and pure seeds of the PPO herbicide tolerant line.

The present invention generally provides methods for producing PPO tolerant seeds. Such methods comprise the following steps: a) producing PPO tolerant plants by transforming plants with a heterologous polynucleotide encoding a PPO b) crossing two plants by pollinating a recipient plant with pollen from a donor plant, wherein at least one of the two plants is tolerant to PPO herbicide c) allowing the pollinated plant to set seeds d) harvesting said seeds; and, optionally e) testing for PPO tolerance by applying a seed treatment comprising a PPO herbicide to seeds resulting from each cross.

These methods are particularly beneficial in the various steps leading to a commercial variety, such as in breeding programs and in the seeds production process. For example, the present invention is used to introgress a PPO herbicide tolerance trait and, optionally, to also introgress another trait that co-segregates with the PPO herbicide tolerance trait, into a particular line, such as an elite inbred line, e.g. an inbred maize line or a soybean cultivar. The introgression of PPO herbicide tolerance into the elite line is for example achieved by recurrent selection breeding, for example by backcrossing. In this case, the elite line (recurrent parent) is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate PPO herbicide tolerance trait. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for PPO herbicide tolerance. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for PPO herbicide tolerance, the progeny is heterozygous for the locus harboring PPO herbicide tolerance, but is like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1 : Theory and Technique, 360-376, incorporated herein by reference). Selection for PPO herbicide tolerance after each cross is carried out using the present invention, i.e. by applying a seed treatment comprising a PPO herbicide to seeds resulting from each cross. PPO herbicide sensitive seeds do not grow and only PPO herbicide tolerant plants are further used in the backcrossing program. In a preferred embodiment, the present invention therefore encompasses methods of breeding a PPO herbicide tolerance trait into an elite line using the teachings of the present invention and comprising the following steps: crossing a first plant with a second plant, wherein one of the plants is tolerant to a PPO herbicide, harvesting the seeds resulting from the cross, applying to said seeds a seed treatment comprising an amount of a PPO herbicide effective to inhibit the eariy growth of a seed sensitive to said PPO tierbicide, placing said seeds under conditions sufficient for germination, and selecting plants that grow, wherein said plants that grow comprise the PPO herbicide tolerance trait.

The present invention is also conveniently used in the production of stable homogenous inbred lines or cultivars (also sometimes called varieties), whereby a particular line is self- pollinated until satisfactory purity and homogeneity of the line is reached. The present invention is similarly used for the commercial production of seeds of a particular inbred line or cultivar. Here again, after each cross a seed treatment of the present invention is applied to the seeds resulting from the cross and only PPO herbicide tolerant plants are selected. In a preferred embodiment, the present invention therefore encompasses methods of producing PPO herbicide tolerant seeds using the teachings of the present invention and comprising the following steps: crossing a PPO tolerant plant with itself, harvesting the seeds resulting from the cross and applying to said seeds a seed treatment comprising an amount of a PPO herbicide effective to inhibit the eariy growth of a seed sensitive to said PPO herbicide. In a preferred embodiment, such seeds are placed under conditions sufficient for germination, and plants that grow are selected, wherein said plants that grow comprise the PPO herbicide tolerance trait.

Similarly, the present invention is used in hybrid seed production. In this case, the present invention is used to assure that all hybrid seeds that germinate and grow in the field are PPO herbicide tolerant. In a preferred embodiment, the present invention therefore encompasses methods producing PPO herbicide tolerance seeds using the teachings of the present invention and comprising the following steps: crossing a first plant with a second plant, wherein one of the plants is tolerant to a PPO herbicide, harvesting the seeds resulting from the cross and applying to said seeds a seed treatment comprising an amount of a PPO herbicide effective to inhibit the early growth of a seed sensitive to said PPO herbicide. In a preferred embodiment, such seeds are placed under conditions sufficient for germination, and plants that grow are selected, wherein said plants that grow comprise the PPO herbicide tolerance trait.

The present invention thus provides a significant advancement to commercial breeding and seeds production processes using PPO herbicide tolerance. Using the present invention large commercial quantities of PPO herbicide tolerant seeds are produced with low impact on the environment, reduced cost and with higher quality control. The present invention is thus a substantially better method as compared to conventional methods using a pre- or post-emergence application of PPO herbicide (see example 8).

The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Example 1 : Soaking seeds in PPO herbicide

Com seeds are soaked in a CGA-276 854 solution.

Example 2: Coating seeds with a PPO herbicide

Com seeds are coated with a slurry of CGA-276 85425WP (WP= wettable powder). The appropriate amount of CGA-276 854 25WP is weighed in glass beakers and made into a slurry by adding a small amount of water. PPO herbicide tolerant and PPO herbicide sensitive com seeds are then placed in separate beakers containing the herbicide slurry. The seeds are stirred in the beakers with a glass rod to get a thorough coating with the herbicide. The seeds are allowed to air dry at room temperature (usually within 2-4 hours).

Example 3: Seed treatment further comprising MaximXL^®

MaximXL^® (fludioxonil + mefenoxam) is mixed with CGA-276854, for example with CGA- 27685425WP, and the resulting mixture is applied as a flowable treatment on com seeds.

Example 4: Seed treatment further comprising Apron^®

Apron^® (metalaxyl) is mixed with CGA-276854, for example with CGA-27685425WP, and the resulting mixture is applied as a flowable treatment on com seeds. Example 5: Application of a seed coating

A protectant coating is applied to treated seeds of examples 1 -4 by impregnating propagation material with a liquid formulation.

A protectant coatings is applied to treated seeds of examples 1 -4 by coating with a combined wet or dry formulation.

Example 6: Planting of seeds in the greenhouse

Dried com seeds are planted in pots containing soil. The soil is a mixture of 37.5% Illinois silty clay loam (5% organic matter), 50% Vero sand (less than 1% organic matter), and 12.5% Promix. The pots are watered as needed for normal plant growth.

Example 7: CGA-276 854 as seed treatment

PPO herbicide tolerant and PPO herbicide sensitive com seeds are coated with different rates of PPO herbicide. The effect of the PPO herbicide of both types of seeds is then determined. The results are shown below:

Rate % PPO herbicide % PPO herbicide

(g ai/ 100 kg sensitive plants with tolerant plants with seed) inhibited early growth inhibited eariy growth vs. total plants vs. total plants

2 82 0

5 95 0

10 100 0

20 100 0

50 100 65

This demonstrates that a PPO herbicide applied as a seed treatment as described in the present invention is effective for selecting PPO herbicide tolerant plants.

Example 8: Comparison of different types of CGA-276 854 application

CGA-276854 is applied as seed treatment (seed soaking or seed coating) or applied on the soil surface (pre-emergence application). The effect of CGA-276 854 is measured as a percentage of PPO herbicide sensitive com plants with inhibited eariy growth vs. total plants.

This demonstrates that a PPO herbicide applied as a seed treatment as described in the present invention is more effective for selecting PPO herbicide tolerant plants than a pre- emergence application of the herbicide.

Claims

What is claimed is:

1. A method for selecting plants tolerant to a PPO herbicide comprising: a) applying to a population of seeds a seed treatment comprising an amount of a PPO herbicide effective to inhibit the eariy growth of a seed sensitive to said PPO herbicide, b) placing said population of seeds under conditions sufficient for germination, and c) selecting plants that grow.

2. A method according to claim 1 , wherein said population of seeds is obtained from a cross, wherein one of the parents in the cross is a PPO herbicide tolerant plant.

3. A method according to claim 1 , wherein said population of seeds is obtained from the self-pollination of a PPO herbicide tolerant plant.

4. A method according to claim 1 , wherein said seed tolerant to a PPO herbicide comprises a heterologous polynucleotide encoding a PPO.

5. A method according to claim 4, wherein said heterologous polynucleotide is under the control of a promoter capable of directing the expression of said heterologous polynucleotide in said seed tolerant to a PPO herbicide or in emerging seedlings derived from said seed tolerant to a PPO herbicide.

6. A method according to claim 5, wherein said heterologous polynucleotide encodes an PPO herbicide tolerant PPO.

7. A method according to claim 6, wherein said heterologous polynucleotide is derived from a bacterium.

8. A method according to claim 6, wherein said heterologous polynucleotide encodes a modified PPO.

9. A method according to claim 8, wherein said heterologous polynucleotide that encodes a modified PPO is derived from a plant.

10. A method according to claim 9, wherein said heterologous polynucleotide that encodes a modified PPO is derived from Arabidopsis.

11. A method according to claim 1 , wherein said population of seeds is derived from a monocotyledonous plant.

12. A method according to claim 11 , wherein said monocotyledonous plant is selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice and barley.

13. A method according to claim 1 , wherein said population of seeds is derived from a dicotyledonous plant.

14. A method according to claim 13, wherein said dicotyledonous plant is selected from the group consisting of soybean, cotton, tobacco, sugar beet and oilseed rape.

15. A method according to claim 1 , wherein said step of applying to said population of seeds a seed treatment comprises soaking said population of seeds in a composition comprising a PPO herbicide.

16. A method according to claim 1 , wherein said step of applying to said population of seeds a seed treatment comprises coating seeds of said population with a composition comprising a PPO herbicide.

17. A method according to claim 1 , wherein said PPO herbicide is selected from the group consisting of an aryluracil, a diphenylether, an oxadiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole and an other heterocyclyl pyrazole derivative, a pyridyl amide derivative, a 3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, a phenopylate and O-phenylpyrrolidino- and O-phenylpiperidinocarbamate analogs of said phenopylate.

18. A method according to claim 1 , wherein said PPO herbicide is a N-Phenyl-uracil.

19. A method according to claim 18, wherein said N-Phenyl-uracil is benzoic acid 2- chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1 (2H)-pyrimidinyl]- 1 ,1 -dimethyl- 2-oxo-2-(2-propenyloxy)ethyl ester (9CI).

20. A treated seed comprising an amount of a PPO herbicide effective to inhibit the early growth of a PPO herbicide sensitive seed.

21. A treated seed according to claim 20, wherein said seed is derived from a monocotyledonous plant.

22 A treated seed according to claim 21 , wherein said monocotyledonous plant is selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice and barley.

23 A treated seed according to claim 20, wherein said seed is derived from a dicotyledonous plant.

24. A treated seed according to claim 23, wherein said dicotyledonous plant is selected from the group consisting of soybean, cotton, tobacco, sugar beet and oilseed rape.

25. A composition comprising an amount of a PPO herbicide effective to inhibit the eariy growth of a PPO herbicide sensitive seed, wherein said composition is applied to a seed as a seed treatment.

26. A composition according to claim 25, wherein said PPO herbicide is selected from the group consisting of an aryluracil, a diphenylether, an oxadiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole and an other heterocyclyl pyrazole derivative, a pyridyl amide derivative, a 3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, a phenopylate and O- phenylpyrrolidino- and O-phenylpiperidinocarbamate analogs of said phenopylate.

27. A composition according to claim 26, wherein said PPO herbicide is a N-Phenyl-uracil.

28. A composition according to claim 27, wherein said N-Phenyl-uracil is benzoic acid 2- chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1 (2H)-pyrimidinyl]-1 ,1 -dimethyl- 2-oxo-2-(2-propenyloxy)ethyl ester (9CI).

29. A composition according to claim 25, wherein said amount of a PPO herbicide is between 1 and 160 g ai/ kg seed.

30. A composition according to claim 29, wherein said amount of a PPO herbicide is between 2 and 40 g ai/ kg seed.

31. A composition according to claim 30, wherein said amount of a PPO herbicide is between 5 and 20 g ai/ kg seed.

32. A method comprising applying to a seed a composition according to claim 25.

33. A method for producing PPO tolerant seeds comprising a) producing PPO tolerant plants by transforming plants with a heterologous polynucleotide encoding a PPO b) crossing two plants by pollinating a recipient plant with pollen from a donor plant, wherein at least one of the two plants is tolerant to PPO herbicide c) allowing the pollinated plant to set seeds d) harvesting said seeds; and, optionally e) testing for PPO tolerance by applying a seed treatment comprising a PPO herbicide to seeds resulting from each cross.

34. The method according to claim 33, wherein the donor plant and the recipient plant are identical.

35. The method according to claim 33, wherein the donor and/or the recipient plant are from an elite line or from a line for inbred or hybrid seed production.