JP2002511274A - Methods for producing plants that are resistant or resistant to herbicides - Google Patents

Abstract

  (57) [Summary] A method for producing a plant that is resistant or resistant to a herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (4HPPD) in vitro, comprising: (i) converting a plant material to a region encoding phytoene desaturase; (Ii) regenerating the transformed material into a morphologically normal plant. In a preferred embodiment, the region contained in the polynucleotide is the sequence represented by SEQ ID No: 1, or at 55 and 60 ° C in 0.3 strength citrate buffered saline containing 0.1% SDS. After rinsing at the same temperature in 0.3 strength citrate buffered saline containing 0.1% SDS, it still hybridizes with the sequence represented by SEQ ID No: 1. It is a sequence complementary to the sequence to be performed.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, inter alia, to tolerant or resistant herbicides.
The present invention relates to a method of producing plants that are stable, and in particular to the production of transgenic plants that exhibit substantial or substantial resistance to herbicides when compared to non-transgenic-like plants.

[0002] Plants that are substantially "tolerant" to a herbicide are dosed to the right when exposed to the agent compared to those provided by similarly exposed non-tolerant-like plants. / Response curves are provided. These dose / response curves plot "dose" on the x-axis and "kill percentage", "herbicidal effect" etc. on the y-axis. Tolerant plants will require more herbicide than non-tolerant-like plants to produce a defined herbicidal effect. Plants that are substantially "resistant" to herbicides, if any, when exposed to herbicides at concentrations and rates typically used in the pesticide community to remove farm weeds. It does not show necrotic, lytic, chlorotic damage or other damage. Plants that are resistant to the herbicide are also resistant to the herbicide. The terms "resistant" and "resistant" should be considered "resistant and / or resistant" within the context of the present application.

A particularly suitable herbicide for the present invention is 4-hydroxy-phenyl in vitro.
Inhibit pyruvate dioxygenase (HPPD or 4-HPPD) enzyme
And what is possible. Such herbicides have been disclosed, for example, especially
The isoforms described in Lance patent applications 95 06800 and 95 13570
Xazoles, and in particular isoxaflutoe, a selective corn herbicide
As described in European Patent Applications 0 496 630, 0 496 631
Any diketonitrile, especially 2-cyano-3-cyclopropyl-1- (2-SO _Two CH_Three-4-CF_Three-Phenyl) propane-1,3-dione and 2-cyano-
3-cyclopropyl-1- (2-SO_TwoCH_Three−4-2,3Cl_TwoPhenyl) pro
Pan-1,3-dione, European Patent Applications 0 625 505 and 0 625 5
08, particularly sulcotrione and mesotrione (BSI-
Presentation), pyrazolinates and pyrazoxifen. Resistant to these herbicides
Are known genes that can provide the HPPD enzyme
.

In accordance with the present invention, there is provided a method for producing a plant that is resistant or resistant to a herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (4HPPD) in vitro, comprising: (i) Transforming a polynucleotide comprising a region encoding phytoene desaturase (PDS); (ii) regenerating the transformed material into a morphologically normal plant. .

[0005] The region contained in the polynucleotide may have the sequence represented by SEQ ID No: 1, or may be expressed in a 0.3 strength citrate buffered saline solution containing 0.1% SDS at 55%. And after incubation at a temperature between 60 ° C. and 60 ° C.
A sequence complementary to the sequence that hybridizes with the sequence represented by SEQ ID No. 1 when rinsed at the same temperature in 0.3-strength citrate-buffered saline containing% SDS . Phytoene desaturase has SEQ ID
No. 1 is preferably of bacterial origin and is derived from Erwinia uredovora and / or especially does not require plastoquinone 9 as a cofactor. However, the desaturases are of plant origin, such as, in particular, monocotyledonous or dicotyledonous plants, in particular Arabidopsis or Arabidopsis or Umbellifere.
rae), for example carrot (Daucus carrotta)
May be used. The desaturase, herbicides may be a naturally occurring, or may be mutated, but at the same time essentially, HPPD inhibitors, for example isoxazole family of herbicides, e.g. Balance ^TM herbicide or triketones Retains resistance characteristics. Herbicide-resistant plants produced by the methods described above can be used in vitro to resist 4HPPD-inhibiting herbicides.
You may choose. However, it is further preferred that the polynucleotide encoding the phytoene desaturase further comprises a selectable marker gene that facilitates the selection of regenerating transformants. Suitable selectable marker genes include: resistance to antibiotics such as kanamycin, hygromycin and gentamicin; resistance to additional herbicides, such as glyphosate-based herbicides; resistance to toxins, such as utilipin.

[0006] Other forms of selection are also available, for example, Hiroyasu Ebin
uma et al. 1997. PNAS Vol. There is a hormone-based selection system such as the multiple automated transformation (MAT) system of 94 pp2117-2121; a visual selection system using the known green fluorescent protein, β-glucuronidase, mannose isomerase, xylose isomerase and 2-DOG.

The plant material is a polynucleotide comprising a region encoding a protein capable of providing resistance or resistance to herbicides, insects, drought and / or fungal, bacterial or viral infections, Alternatively, the polynucleotide may be further transformed with a polynucleotide capable of encoding a protein that provides improved quality attributes, such as increased yield, altered starch quality, and / or increased nutrient content, or It may have been converted.

[0008] Sequences within the polynucleotide that encode the protein are linked to promoters and terminators that can function in plants. Such promoters and terminators are not closely related to the present invention per se, are well known to those skilled in the art, and include, for example, CaMV35S, FMV35S, NOS, OCS and E9 (
Promoter and terminator (from the small subunit of RUBISCO) or the promoter and terminator of the alpha-tubulin gene (EP
-A 652, 286). Preferably, the aid is obtained of a promoter control sequence that supports the overexpression of the coding sequence, for example one containing at least one histone promoter as described in EP-A-507,698.

[0009] In accordance with the present invention, other control sequences associated with the promoter control sequence and located between the promoter and the coding sequence, such as transcription or translation enhancers, such as international patent applications, PCT publications incorporated herein by reference. WO 87/07644
Tobacco etch vir as described in
us) (TEV) formed by a part of the sequence of the N-terminal mature part of a plant gene encoding a translation activator, or a plastid localizing enzyme, as described in EP-A-508,909. Single or two transit peptides and, in the case of two transit peptides, those which may be separated by intervening sequences, ie, in the direction of transcription, the transport of the plant gene encoding the plastid localizing enzyme A sequence encoding the peptide, a part of the sequence of the N-terminal mature part of the plant gene encoding the plastid localizing enzyme, and then a sequence encoding the second transit peptide of the plant gene encoding the plastid localizing enzyme. It is equally possible to use those that include.

The plant material is a polynucleotide comprising a region encoding a protein capable of providing the plant with resistance or resistance to herbicides, insects, drought and / or fungal, bacterial or viral infections, or May have been further transformed with a polynucleotide capable of encoding a protein that provides improved quality attributes, such as increased yield, altered starch quality, and / or increased nutrient content, or And may be transformed.

[0011] Proteins that can provide herbicide resistance include glyphosate oxidoreductase (GOX), 5-enol-pyruvyl-3-phosphoshikimate synthetase (
EPSPS), phosphinothricin acetyltransferase (PAT)
, Hydroxyphenylpyruvate dioxygenase (HPPD), glutathione S-transferase (GST), cytochrome P450, acetyl-COA carboxylase (ACCase), acetolactate synthase (ALS), protoporphyrinogen oxidase (PROTOX), dihydropteroic acid Synthase,
Polyamine transporter protein, superoxide dismutase (SOD), bromoxynil nitrilase, tf available from Alcaligenes eutrophus
The product may be selected from the group consisting of a product of the dA gene and a known mutagenized or otherwise modified variant of the protein.

As indicated above, polynucleotides that may transform plant material include:
i) converting the translation product of the region into a plastid such as chloroplast or mitochondria;
Peptides that can be targeted to other organelles or plant cell walls; and / or
Or (ii) 5 'of the protein coding region, which codes for an untranslated translation-enhancing sequence
May be included.

[0013] The polynucleotide may be codon-optimized, or may be modified so that at least transcription is enhanced once the polynucleotide is incorporated into plant material. Therefore, the polynucleotide used to transform the material is mR
The plant is modified so that the NA-labile coding motif and / or the incidental splicing region is removed, or uses a plant-preferred codon, and the expression in the plant of the modified polynucleotide is such that the unmodified polynucleotide The protein coding region may be modified to yield a substantially similar protein having substantially the same activity / function as obtained by unmodified polynucleotide expression in a plant in which it is endogenous. However, with respect to the herbicide resistance-conferring region, when the modified polynucleotide comprises a plant-preferred codon, the protein-coding region in the modified polynucleotide and endogenously included in the plant, and substantially Identical to a comparable protein coding region that encodes the same protein The degree of less than about 70%.

[0014] Transformation techniques are well known and particle mediated biolistics.
Transformation, Agrobacterium-mediated transformation,
Protoplast transformation (optionally in the presence of polyethylene glycol); sonication of plant tissue, cells or protoplasts in a medium containing polynucleotides or vectors; micro-insertion of polynucleotides or vectors into totipotent plant material ( Optionally known silicon carbide "whiskers"
)) Technology), electroporation and the like.

[0015] The invention further relates to whole morphologically normal fertile (or male sterile) plants, and the progeny of such plants, such plants and progeny, regenerated from the material referred to in the paragraph immediately preceding the preceding paragraph. And some of such plants and progeny. The plants of the present invention to be transformed include small grain cereals, oilseed crops, fiber plants (fib
replant), fruits, vegetables, plantation crops and trees. Particularly preferred such plants include soybean, cotton, tobacco,
Sugar beet (sugarbeet), oilseed rape, canola (canola), flax (flax), sunflower (sunflower),
Potato (potato), tomato (tomato), purple coconut palm (
alfalfa), lettuce, corn, wheat (whe)
at), sorghum, rye, banana, barley, oat, oat, turf grass, forage glass, sugar cane, pea (pea) field bean, rice, rice, pine, poplar, apple, grape (g)
rape, citrus and nut plants.

The transformed plants according to the invention are resistant or resistant to certain herbicides, such herbicides being described, for example, in particular in French patent applications 95 06800 and 95 135
Isoxazoles described in No. 70, and in particular 4- [4-CF3-2- (methylsulfonyl) benzoyl] -5-cyclopropylisoxazole, and in particular isoxaflutole, a selective corn herbicide , EP-A-49
Jiketonitoriru such as those described in 6,630 and EP-A-496,631, especially _{2-cyano-3-cyclopropyl--1- (2-SO 2 CH 3} -4-C
F ₃ - phenyl) propane-1,3-dione and _{2-cyano-3-cyclopropyl--1- (2-SO 2 CH 3} -4-2,3Cl 2 phenyl) propane-1,3
Diones, the triketones described in EP-A-625,505 and EP-A-625,508, in particular sulcotrione, mesotrione (BSI-presented), pyrazolinates and pyrazoxifenes.

The present invention further provides whole morphologically normal fertile (or male sterile) plants, progeny of such plants, seeds of such plants and progeny, and seeds of such plants and progeny, resulting from the methods of the present invention. Including some.

The invention further provides the use of a polynucleotide comprising a region encoding a phytoene desaturase in the production of a plant material that is resistant or resistant to a herbicide that inhibits the enzyme 4-HPPD in vitro.

The present invention is further directed to a method of selectively regulating weeds on a farm, wherein the farm comprises weeds and crop plants, wherein the farm is capable of inhibiting the enzyme 4-HPPD in vitro. Providing said method, comprising applying a herbicide, wherein said plant has been transformed with a coding region of a polynucleotide comprising a sequence encoding a phytoene desaturase and characterized by expressing said coding region. I do.

The phytoene desaturase coding sequence is that represented in SEQ ID No: 1 or between 55 and 60 ° C. in 0.3 strength citrate buffered saline containing 0.1% SDS. After incubation at a temperature of 0.1
It is particularly preferable that the DNA sequence is complementary to a sequence that hybridizes with the sequence represented by SEQ ID No. 1 when rinsed at the same temperature in a 0.3-strength citrate buffered saline solution containing% SDS. . The herbicide may be selected from the group consisting of mesotrione (BSI-presented), pyrazolinates and pyrazoxifen, Balance ^™ , sulcotrione and the like. The farm may be treated with an insecticide selected from the group consisting of fungicides, insecticides and nematicides either before or after applying the herbicide to the farm.

The present invention will now be described through the following non-limiting examples, figures and sequence listings. Where: SEQ ID No: 1 is the sequence of the phytoene desaturase (dehydrogenase) gene isolated from Erwinia uredobora. Those skilled in the art will appreciate that any phytoene-
It will be appreciated that the desaturase gene may be used.

SEQ ID No: 2 is a protein encoded by SEQ ID No: 1. SEQ ID No: 3 is a polynucleotide sequence encoding a pea rubisco small subunit transit peptide.

[0023] SEQ ID No: 4 is the amino acid sequence encoded by SEQ ID No: 3.

[0024]

EXAMPLES in vitro enzyme 4-HPPD production of resistant der Ru plant herbicides capable of inhibiting the PDS gene (crtI) non-green phytopathogenic bacterial rot, and cloned from Erwinia uredovora, And a plasmid (pYPIET4) containing the CaMV 35S promoter and the chloroplast transit peptide (Misawa et al.,
1993) was overexpressed in transgenic tobacco and tomato. c
Homozygous tomato lines overexpressing the rtI gene were obtained as well as tobacco plants containing the same construct.

Construction of plasmid pYPIET4 with tp-crtI gene Using recombinant standard techniques, recombinant DNA technology was performed. Pea ribulose-1,5
-The DNA sequence encoding the transit peptide (TP) in the precursor of the bisphosphate carboxylase (Rubisco) small subunit has a 204 bp HindII
Plasmid pSNIF83 (Schreier et al.,
1985) and its Spg1 site contains a tp processing site. The intact phytoene desaturase gene of Erwinia uredobora (
plasmid (CrtI) (Fraser et al. (1992) J. Mol.
Biol. Chem 267 19891-1895) was digested with BamHI and HindIII, and the 1.57 kb BamHI-HindIII fragment with the truncated crtI gene was isolated. The above 204 bp HindIII-Sph1 TP fragment was converted into a 76 bp synthetic fragment having an open reading frame from the cohesive end of the Sph1 site including the crtI initiation codon to the cohesive end of the BamHI site, and a 1.57 kb BamHI-HindIII fragment. Connected to. The desired 1.84 kb HindIII fragment carrying the tp-crt1 chimeric gene was isolated, filled in with Klenow enzyme, and the binary vector b
B-121 (purchased from Clontech laboratories) to β-
Sma1-Sa fragment of the 10.9 kb fragment from which the glucuronidase gene has been removed
Ligation was performed within the c1 site. The desired plasmid pYPEIT4 thus generated
Is shown in FIG. The start codon of the transit peptide and the intact Crt1 are underlined. This HindIII fragment carrying the tp-crtI gene is surrounded by the CaMV 35S promoter and NOS terminator of the binary vector pB1121 to direct sufficient expression in tissues of transgenic tobacco and tomato plants. As a control, without tp, CaMV 35S
Intact cr surrounded by promoter and NOS terminator
A plasmid pBICAR4 having the t1 gene was constructed. Plasmid pYPEIT4 was introduced into tobacco and tomato material by known techniques, and then the material was again regenerated into intact plants by known technology.

[0026] In crtI genes for mesotrione and Isoki Safurutoru tomato plants that are transformed resistant "wild-type" derived (i.e. untransformed) tomato plants cv. Ailsa C
raig homozygous seed and a plant transformed with the crtI gene from Erwinia uredbora (see above) are sown on a peat-based compost in a 3 inch flowerpot and Transferred to greenhouse. The plants are grown under a 16-hour photoperiod at a temperature of 20 degrees C / day / 16 degrees C at night for about 4 weeks, after which four replicates with mesotrione or isoxaflutol (Balance ^™ herbicide) at the three leaf stage. It was processed after emergence. The chemicals are suspended in water and, as shown in Table 1, at a rate in the range of 1 to 500 grams of active ingredient per hectare (g active ingredient / ha) at a spray volume of 200 liters per hectare. , Applied via a track sprayer.
The plants were grown for an additional 25 days and then visually assessed for herbicidal damage compared to untreated "control" plants. Typical phytotoxic symptoms observed were extreme bleaching / bleaching and necrosis of leaves and new growths. The results of this test are shown in Table 1 below, where the "% damage / phytotoxicity" score represents the average of the visual assessment of each of the four treated replicates.

[0027]

[Table 1]

As can be seen, expressing bacterial PDS from Erwinia uredobora,
Plants transformed with the crtI gene show enhanced resistance to mesotrione and isoxaflutol compared to wild-type, untransformed tomatoes. For example, 11 g active ingredient / ha of mesotrione caused 50% phytotoxicity in wild-type tomatoes, while only 9% damage was observed in transformed plants. Similarly, wild-type plants have 500 g active ingredient / h compared to those containing the crtI gene.
The isoxaflutole in a is significantly more damaged.

Those skilled in the art will recognize that the present invention is not limited to the above. For example, plants other than tomato and tobacco may be transformed with a gene encoding a PDS enzyme, whether from a bacterial source or otherwise.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the structure of plasmid pYPEIT4 having the Erwinia uredobola crtI gene containing the pea rubisco small subunit transit peptide sequence (designated TP).

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (71) Applicant Fernhurst, Haslemere, Surrey GU27 3JE, United Kingdom (72) Inventor Brian, Ian Bennett Berkshire Arlsey 426, UK England, Bracknell, Jerotz Hill Research Station F-term (reference) 2B030 AA02 AA03 AB03 AD05 CA06 CA17 CA19 CB02 CD21 4B024 AA08 BA07 CA04 DA01 EA04 GA14 HA01

Claims (21)

[Claims] 1. A method for producing a plant that is resistant or resistant to a herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (4HPPD) in vitro, comprising: (i) converting a plant material to phytoene desaturase; (Ii) transforming the material thus transformed into morphologically normal plants and transforming 4HPPD in vitro from a population of regenerated products. Selecting a plant that is resistant or resistant to the herbicide to be inhibited. 2. The method according to claim 1, wherein the region contained in the polynucleotide is SEQ ID No.
After incubation at a temperature between 55 and 60 ° C. in 0.3 strength citrate buffered saline with the sequence represented by 1 or with 0.1% SDS, 0.1% SDS was The method according to claim 1, which is a sequence complementary to a sequence that hybridizes with a sequence represented by SEQ ID No. 1 when rinsed at the same temperature in a citrate buffered saline having 0.3 strength. . 3. The method of claim 1, wherein the phytoene desaturase is of plant origin.
the method of. 4. The method of claim 1, wherein the phytoene desaturase is of bacterial origin.
the method of. 5. The method of claim 4, wherein the phytoene desaturase is isolatable from Erwinia uredovora. 6. The method of any one of claims 1 to 5, wherein the polynucleotide further comprises a selectable marker gene. 7. The selectable marker gene comprises an antibiotic resistance, a herbicide resistance, a toxin resistance, a nutritional marker, a visual marker, and a marker gene used in a hormone-based selection system. Selected from a group,
The method of claim 6. 8. A plant material comprising: a herbicide, an insect, a dried and / or dried herbaceous material.
Or a polynucleotide comprising a region encoding a protein capable of providing resistance or resistance to fungal, bacterial or viral infection, or increased yield,
2. The polynucleotide of claim 1, further modified or transformed with a polynucleotide capable of encoding a protein that provides improved quality attributes such as altered starch quality and / or increased nutrient content. Any one of or seven
One way. 9. The sequence in a polynucleotide which encodes a protein,
2. The method according to claim 1, which is linked to a promoter and a terminator operable in a plant.
The method may be any one of -8. 10. Proteins capable of providing herbicide resistance include glyphosate oxidoreductase (GOX), 5-enol-pyruvyl-phosphoshikimate synthetase (EPSPS), phosphinothricin acetyltransferase (PAT) ), Hydroxyphenylpyruvate dioxygenase (HPPD)
), Glutathione S transferase (GST), cytochrome P450, acetyl-COA carboxylase (ACCase), acetolactate synthase (AL
S), protoporphyrinogen oxidase (PROTOX), dihydropteroate synthase, polyamine transport protein, superoxide dismutase (SOD), bromoxynil nitrilase, alkalinegenes eutrophus (
9. The product of the tfdA gene available from Alcaligenes eutrophus, selected from the group consisting of farnesyl pyrophosphate synthase and known mutagenesis or otherwise modified variants of said protein. Either method of 9. 11. The polynucleotide, wherein the sequence encoding the protein is:
(I) a peptide capable of targeting the translation product of the region to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls; and / or (ii) encoding a non-translationally enhanced translational sequence. 11. The method according to any one of claims 1 to 10, comprising a 5 'region. 12. The polynucleotide used to transform a material, wherein the polynucleotide is m-type.
Either the RNA-labile coding motif and / or the spontaneous splicing region has been modified to be removed or, alternatively, the plant uses codons that are preferred for plant expression of the unmodified polynucleotide. The protein coding region may be modified to yield a substantially similar protein having substantially the same activity / function as obtained by unmodified polynucleotide expression in a plant in which it is endogenous. However, with respect to the herbicide resistance-conferring region, when the modified polynucleotide comprises a plant-preferred codon, the protein-coding region in the modified polynucleotide and endogenously included in the plant, and substantially Same as a comparable protein coding region encoding the same protein Degree of sex, less than about 70%, claims 1 11
Any one of the methods. 13. The method according to claim 1, wherein the 4-HPPD-inhibiting herbicide is isoxaflutol, a diketonitrile, for example, 2-cyano-3-cyclopropyl-1- (2-SO ₂ CH ₃ -4-CF ₃ -phenyl) propane-. 1,3-dione and _{2-cyano-3-cyclopropyl--1- (2-SO 2 CH 3} -4-2,3Cl 2 phenyl) propane -
1,3-dione, triketones such as sulcotrione and mesotrione (
13. A method according to any one of claims 1 to 12, wherein the method is selected from the group consisting of BSI-presentation), pyrazolinate and pyrazoxifene. 14. The method according to any one of claims 1 to 13, wherein the herbicide is applied after germination. 15. A morphologically normal fertile (or male sterile) whole plant, progeny of such a plant, resulting from the method according to any one of claims 1 to 14,
Seeds of such plants and progeny, and parts of such plants and progeny. 16. The plant of claim 15, wherein the plant is selected from the group consisting of banana, cotton, corn, maize, tomato, and vines. 17. Use of a polynucleotide comprising a region encoding a phytoene desaturase in the production of a plant material that is resistant or resistant to a herbicide that inhibits the enzyme 4-HPPD in vitro. 18. A method for selectively controlling weeds on a farm containing weeds and crop plants, comprising applying a herbicide capable of inhibiting the enzyme 4-HPPD in vitro to the farm. Wherein the plant has been transformed with a polynucleotide coding region comprising a sequence encoding a phytoene desaturase,
And said method is characterized by expressing said coding region. 19. The polynucleotide according to any one of claims 2 to 12, wherein the polynucleotide is
19. The method of claim 18, wherein the method is referred to in any of the following claims. 20. The herbicide according to claim 1, wherein the herbicide is isoxaflutol, a diketonitrile, for example, 2-cyano-3-cyclopropyl-1- (2-SO ₂ CH ₃ -4-CF _3.
- phenyl) propane-1,3-dione and _{2-cyano-3-cyclopropyl--1- (2-SO 2 CH 3} -4-2,3Cl 2 phenyl) propane-1,3-dione, triketones, e.g. 20. The method of any of claims 18 or 19, wherein the method is selected from the group consisting of sulcotrione, and mesotrione (BSI-presented), pyrazolinate, and pyrazoxifene. 21. The treatment of the farm with an insecticide selected from the group consisting of a fungicide, an insecticide and a nematocide, either before or after applying the herbicide to the farm. The method may be any one of 18-20.