METHOD OF PRODUCING PLANTS WHICH ARE TOLERANT OR RESISTANT TO HERBICIDES

Abstract: A method of making plants which are resistant or tolerant to herbicides which, in vitro, inhibit 4-hydroxyphenylpyruvate dioxygenase (4HPPD) comprises the steps of: (i) transforming plant material with a polynucleotide comprising a region encoding a phytoene desaturase; (ii) regenerating the thus transformed material into morphologically normal plants. In a preferred embodiment the region comprised by the polynucleotide is the sequence depicted in SEQ ID No.1, or is a sequence which is complementary to one which when incubated at a temperature of between 55 and 60 DEG C in 0.3 strength citrate buffered saline containing 0.1 % SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 % SDS still hybridises with the sequence depicted in SEQ ID No.1.

Description: - 1 - METHOD OF PRODUCING PLANTS WHICH ARE TOLERANT OR RESISTANT TO HERBICIDES The present invention relates inter alia, to a method of producing plants which are tolerant or resistant to herbicides and in particular to the production of transgenic plants which exhibit substantial resistance or substantial tolerance to herbicides when compared with non transgenic like plants. Plants which are substantially "tolerant" to a herbicide when they are subjected to it provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non tolerant like plants. Such dose/response curves have "dose" plotted on the x-axis and "percentage kill", "herbicidal effect" etc. plotted on the y-axis. Tolerant plants will require more herbicide than non tolerant like plants in order to produce a given herbicidal effect. Plants which are substantially "resistant" to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the herbicide at concentrations and rates which are typically employed by the agrochemical community to kill weeds in the field. Plants which are resistant to a herbicide are also tolerant of the herbicide. The terms "resistant" and "tolerant" are to be construed as "tolerant and or resistant" within the context of the present application. The herbicides of particular relevance to the present invention are those which are capable in vitro of inhibiting 4-Hydroxy-phenylpyruvate dioxygenase (HPPD or 4HPPD) enzymes. Such herbicides have been disclosed, such as the isoxazoles described especially in the French Patent Applications 95 06800 and 95 13570 and especially isoxaflutole, a selective maize herbicide, diketonitriles such as those described in European Applications 0 496 630, 0496 631, in particular 2-cyano-3-cyclopropyl-l-(2-SO 2 CH 3 -4-CF 3 - phenyl)propane-l,3-dione and 2-cyano-3-cyclopropyl-l-(2-SO 2 CH 3 -4-2,3Cl 2 phenyl)propane- 1,3-dione, triketones described in European Applications 0 625 505 and 0 625 508, in particular sulcotrione, mesotrione (BSI-proposed), pyrazolynate and pyrazoxyfen. Known genes capable of providing for tolerance to these herbicides are those which encode HPPD enzymes. According to the present invention there is provided a method of making plants which are resistant or tolerant to herbicides which - in vitro - inhibit 4- hydroxyphenylpyruvate dioxygenase (4HPPD) comprising the steps of: (i) transforming plant material with a polynucleotide comprising a region encoding a phytoene desaturase (PDS); (ii) regenerating the thus transformed material into morphologically normal plants. The region comprised by the polynucleotide may have the sequence depicted in SEQ ID No. 1 , or may be a sequence which is complementary to one which when incubated at a temperature of between 55 and 60°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No. 1. It is preferred that the phytoene desaturase is of bacterial origin such as that depicted in SEQ ID No. 1 and being derived from Erwinia uredovora, and/or in particular is one which does not require plastoquinone 9 as a co-factor. The desaturase may, however be of plant origin, such as especially of monocotyledonous or dicotyledonous plants, especially of Arabidopsis or of Umbelliferae, such as, for example, the carrot (Daucus carotta). It can be native or possibly mutated while at the same time fundamentally retaining a property of herbicidal tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family such as the Balance ™ Herbicide or triketones. The herbicide resistant plants produced by the above method may be selected through their resistance to herbicides which in vitro, inhibit 4HPPD. In may however, be further preferred that the polynucleotide encoding the phytoene desaturase further comprises a selectable marker gene to facilitate the selection of regenerated transformats. Suitable selectable marker genes include; resistance to antibiotics such as kanamycin, hygromycin and gentamycin; resistance to iϊirtrier herbicides such as glyphosate based herbicides; resistance to toxins such as eutypine. Other forms of selection are also available such as hormone based selection systems such as the Multi Auto Transformation (MAT) system of Hiroyrasu Ebinuma et al. 1997. PNAS Vol. 94 pp2117-2121; visual selection systems which use the known green flourescence protein, β glucoronidase, mannose isomerase, xylose isomerase and 2-DOG. The plant material may be, or may have been, further transformed with a polynucleotide comprising a region encoding a protein capable of providing the plant material with resistance or tolerance to herbicides, insects, desiccation and/or fungal, bacterial or viral infections, or with a polynucleotide capable of encoding proteins which provide for improved quality traits such as increased yield, altered starch quality and/or increased nutrient content. The protein encoding sequences within the polynucleotide are bounded by plant operable promoters and terminators. Such promoters and terminators, which are per se not germane to the invention, are well known to the skilled man and include, for example, the CaMV35S, FMV35S, NOS, OCS and E9 (derived from the small subunit of RUBISCO) promoters and terminators, or the promoter and terminator of a gene of alpha-tubulin (EP-A 652,286). Preferably, recourse is made to a promoter regulation sequence which favours the over-expression of the coding sequence, such as, for example, that comprising at least one histone promoter such as described in EP-A-507,698. According to the invention, it is equally possible to use, in association with the promoter regulation sequence, other regulation sequences which are situated between the promoter and the coding sequence, such as transcriptional or translational enhancers such as, for example, tobacco etch virus (TEV) translation activator described in International Patent application, PCT publication number WO87/07644 which is incorporated herein by reference, or of transit peptides, either single, or double, and in this case possibly separated by an intermediate sequence, that is to say comprising, in the transcription direction, a sequence coding for a transit peptide of a plant gene coding for a plastid localization enzyme, a part of the sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localization enzyme, formed by a part of the sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, such as described in EP- A-508,909. The plant material may have been, or may subsequently be - further transformed with a polynucleotide comprising a region encoding a protein capable of providing the plant with resistance or tolerance to herbicides, insects, desiccation and/or fungal, bacterial or viral infections, or with a polynucleotide capable of encoding proteins which provide for improved quality traits such as increased yield, altered starch quality and/or increased nutrient content. - 4 - The protein capable of providing for herbicide resistance may be selected from the group consisting of glyphosate oxido-reductase (GOX), 5-enol-pyruvyl-3-phosphoshikimate synthetase (EPSPS), phosphinothricin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-COA carboxylase (ACCase), Acetolactate synthase (ALS), protoporphyrinogen oxidase (PROTOX), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil nitrilase, the product of the tfdA gene obtainable from Alcaligenes eutrophus, and known mutagenised or otherwise modified variants of the said proteins. As indicated above, the polynucleotide with which the plant material may be transformed may comprise 5' of the protein encoding regions regions which encode: (i) a peptide which is capable of targeting the translation products of the regions to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls; and/or (ii) non-translated translational enhancing sequences. The polynucleotide may be codon-optimised, or otherwise altered to enhance at least transcription once it is incorporated into plant material. Thus the polynucleotide used to transform the material may be modified in that mRNA instability encoding motifs and/or fortuitous splice regions may be removed, or plant preferred codons may be used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if - in respect of the herbicide resistance conferring regions - the thus modified polynucleotide comprises plant preferred codons, the degree of identity between the protein encoding regions within the modified polynucleotide and like protein encoding regions endogenously contained within the said plant and encoding substantially the same protein is less than about 70%. Transformation techniques are well known and include particle mediated biolistic transformation, Agrobacterium-mediatQd transformation, protoplast transformation (optionally in the presence of polyethylene glycols); sonication of plant tissues, cells or protoplasts in a medium comprising the polynucleotide or vector; micro-insertion of the polynucleotide or vector into totipotent plant material (optionally employing the known silicon carbide "whiskers" technique), electroporation and the like. - 5 - The invention still further provides morphologically normal fertile (or male sterile) whole plants regenerated from the material mentioned in the paragraph immediately preceding the last and the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny. The transformed inventive plants include small grain cereals, oil seed crops, fibre plants, fruit, vegetables, plantation crops and trees. Particularly preferred such plants include soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, grape, citrus and nut plants. The transformed plants of the invention have tolerance or resistance to certain herbicides such as the isoxazoles described especially in French Patent Applications 9506800 and 95 13570 and especially of 4-[4-CF3-2-(methylsulphonyl)benzoyl]-5- cyclopropylisoxazole, and especially isoxaflutole, a selective maize herbicide, the diketonitriles such as those described in EP-A-496,630 and EP-A-496,631 , in particular 2- cyano-3-cyclopropyl-l-(2-SO 2 CH 3 -4-CF 3 -phenyl)propane-l,3-dione and 2-cyano-3- cyclopropyl-l-(2-SO 2 CH 3 -4-2,3-Cl 2 -phenyl)propane-l,3-dione, and the triketones described in EP-A-625,505 and EP-A-625,508, in particular sulcotrione, mesotrione (BSI-proposed), pyrazolynate and pyrazoxyfen. The invention further includes a morphologically normal fertile (or male sterile) whole plant resulting from the method of the invention, the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny. The invention still further provides the use of a polynucleotide comprising a region encoding a phytoene desaturase in the production of plant material which is resistant or tolerant to herbicides which - in vitro - inhibit the enzyme 4-HPPD. The invention still further provides a method of selectively controlling weeds in a field, the field comprising weeds and crop plants, the method comprising application to the field of a herbicide which - in vitro - is capable of inhibiting the enzyme 4-HPPD, characterised in that the plants have been transformed with and express the coding regions of a polynucleotide comprising a sequence encoding a phytoene desaturase. It is particularly preferred that the phytoene desaturase encoding sequence is that which is depicted in SEQ ID No. 1, or is complementary to one which when incubated at a temperature of between 55 and 60°C in 0.3 strength citrate buffered saline containing 0.1%o - 6 - SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1%) SDS still hybridises with the sequence depicted in SEQ ID No. 1. The herbicide may be selected from the group consisting of mesotrione (BSI-proposed), pyrazolynate and pyrazoxyfen, Balance,™ sulcotrione etc. The field may be treated with a pesticide selected from the group consisting of a fungicide, insecticide and nematicide, either prior to or post application to the field of the herbicide. The invention will now be described by way of the following non-limiting example, figure and the Sequence Listing in which: SEQ ID No. 1 is the sequence of the phytoene desaturase (dehydrogenase) gene isolated from Erwinia uredovora. The person skilled in the art will recognise that any phytoene desaturase gene may be used in the production of plants having resistance/tolerance to the herbicides described above. SEQ ID No. 2 is the protein encoded by SEQ ID No 1. SEQ ID No.3 is the polynucleotide sequence encoding the pea rubisco small subunit transit peptide. SEQ IN No. 4 is the amino acid sequence encoded by SEQ ID No. 3. Figure 1 is the structure of plasmid pYPEIT4 carrying the Erwinia uredovora crtl gene with the transit peptide sequence (depicted as TP) of the pea rubisco small subunit. EXAMPLE Production of plants tolerant to herbicides capable of inhibiting the enzyme 4-HPPD in vitro. The PDS gene (crtl) was cloned from Erwinia uredovora, a non-green phytopathogenic bacterial rot, and over-expressed in transgenic tobacco and tomato using a plasmid containing the CaMV 35S promoter and a chloroplast transit peptide (pYPIET4) (Misawa et al., 1993). Homozygous tomato lines over-expressing the crtl gene were obtained as were tobacco plants containing the same construct. Construction of plasmid pYPIET4 carrying the tp-crtl gene Recombinant DNA techniques were performed using standard methods. A DNA sequence coding for the transit peptide (TP) in the precursor of the ribulose-l,5-bisphosphte carboxylase (Rubisco) small subunit of pea was isolated from plasmid pSNIF83 (Schreier et al, 1985) as a 204 bp Hindlll-Sphl fragment, whose Spgl site contains the tp processing site. Plasmid pCRT-1 (Fraser et al (1992) J.Biol.Chem 267 19891-19895) carrying the intact phytoene desaturase gene (crtl) of Erwinia uredovora was digested with BamHl and Hindlll, and a 1.57 kb BamHl-Hindlll fragment carrying the truncated crtl gene was isolated. The above 204 bp Hindlll-Sphl TP fragment was ligated with a 76bp synthesized fragment which carries the reading frame from the cohesive end for the Sphl site containing the crtl initiation codon to that of the BamHl site, and with the 1.57 kb BamHl-Hindlll fragment. The desired 1.84 kb HinάWl fragment carrying the tp-crtl chimeric gene was isolated, filled in with Klenow enzyme, and ligated into the Smal-Sacl site of a 10.9 kb fragment removing the β-glucuronidase gene from the binary vector bB/121 (purchased from Clontech laboratories). Thus, the desired plasmid pYPEIT4 was created, shown in Figure 1. The initiation codons for the transit peptide and the intact Crtl are underlined. This Hindlll fragment carrying the tp-crtl gene is surrounded by the CaMV 35S promoter and the NOS terminator of the binary vector pB1121 in order to lead to sufficient expression in the tissues of transgenic tobacco and tomato plants. As a control, plasmid pBICAR4 was constructed which carries an intact crtl gene without tp surrounded by the CaMV 35S promoter and the NOS terminator. The plasmid pYPElT4 was introduced into tobacco and tomato material by known techniques and the material then regenerated into intact plants, again by known techniques. Tolerance of Tomato Plants transformed with crtl gene to Mesotrione and Isoxaflutole Homozygous seed of tomato plants cv. Ailsa Craig, derived from 'wild type' (i.e. un- transformed) and plants transformed with the crtl gene from Erwinina uredovora, (see above) were sown in a peat-based compost in 3 inch pots and transferred to the glasshouse. Plants were grown at 20/16 degrees day /night temperature under a 16 hour photoperiod for approximately 4 weeks prior to post-emergence treatment of four replicates with mesotrione or isoxaflutole (Balance ™ Herbicide) at the 3 leaf stage. The chemicals were suspended in water and applied, via a track sprayer at a spray volume of 200 litres per hectare, at rates ranging from 1 to 500 grammes active ingredient per hectare (g a.i./ha), as shown in Table 1. The plants were left to grow for a further 25 days and then assessed visually for herbicidal damage compared to untreated 'control' plants. Typical phytotoxic symptoms observed were extreme chlorosis/bleaching and necrosis of leaves and new growth. The results from this test are given in Table 1 below where the '% Damage/Phytotoxicity' scores represent the mean of the visual assessment from each of the four treatment replicates. - 9 - Table 1 Chemical Rate % Damage/Phytotoxicity (g a.i./ha) (25 days after treatment) Wild Type Transformed (Un-Transformed) (crtl ) Mesotrione 1 25 0 3 29 0 11 50 9 33 81 49 Isoxaflutole 1 11 2 (Balance™ ) 5 15 4 15 19 6 50 21 8 150 34 19 500 65 31 As can be seen, plants transformed with the crtl gene which expresses the bacterial PDS from Erwinia uredovora, demonstrate elevated tolerance to mesotrione and isoxaflutole compared to wild type, un-transformed tomatoes. For example, 11 g a.i./ha of mesotrione caused 50%) phytotoxicity to wild type tomatoes but only 9%> injury is observed in the transformed plants. Similarly, wild type plants are significantly more damaged by 500 g a.i./ha of isoxaflutole than those containing the crtl gene. The skilled man will recognise that the invention is not limited to that described above. For example, plants other than tomato and tobacco may be transformed with a gene encoding a PDS enzyme, whether derived from a bacterial source or otherwise.

Claims: - 10 - CLAIMS 1. A method of making plants which are resistant or tolerant to herbicides which - in vitro - inhibit 4-hydroxyphenylpyruvate dioxygenase (4HPPD) comprising the steps of: (i) transforming plant material with a polynucleotide comprising a region encoding a phytoene desaturase (PDS); (ii) regenerating the thus transformed material into moφhologically normal plants and selecting from the population of regenerants those plants which are resistant or tolerant to herbicides which in vitro inhibit 4HPPD. 2. A method according to claim 1 , wherein the region comprised by the polynucleotide is the sequence depicted in SEQ ID No. 1, or is a sequence which is complementary to one which when incubated at a temperature of between 55 and 60°C in 0.3 strength citrate buffered saline containing 0.1 %> SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 %> SDS still hybridises with the sequence depicted in SEQ ID No. 1. 3. A method according to claim 1 , wherein the phytoene desaturase is of plant origin. 4. A method according to claim 1, wherein the phytoene desaturase is of bacterial origin. 5 A method according to claim 4 wherein the phytoene desaturase is isolatable from Erwinia uredovora. 6. A method according to any one of claims 1 to 5 wherein the polynucleotide further comprises a selectable marker gene. - 1 1 - 7. A method according to claim 6 wherein the said selectable marker gene is selected from the group consisting of antibiotic resistance conferring, herbicide resistance conferring, toxin resistance conferring, nutritional markers, visual markers and marker genes used in hormone based selection systems. 8. A method according to any one of claims 1 to 7, wherein the plant material has been or is further transformed with a polynucleotide comprising a region encoding a protein capable of providing the plant material with resistance or tolerance to herbicides, insects, desiccation and/or fungal, bacterial or viral infections, or with a polynucleotide capable of encoding proteins which provide for improved quality traits such as increased yield, altered starch quality and/or increased nutrient content. 9. A method according to any one of claims 1 to 8, wherein the protein encoding sequences within the polynucleotide are bounded by plant operable promoters and terminators. 10. A method according to either of claims 8 or 9, wherein the protein capable of providing for herbicide resistance is selected from the group consisting of glyphosate oxido-reductase (GOX), 5-enol-pyruvyl-3-phosphoshikimate synthetase (EPSPS), phosphinothricin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-COA carboxylase (ACCase), Acetolactate synthase (ALS), protopoφhyrinogen oxidase (PROTOX), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil nitrilase, the product of the tfdA gene obtainable from Alcaligenes eutrophus, farnesyl pyrophosphate synthase and known mutagenised or otherwise modified variants of the said proteins. - 12 - 1 1. A method according to any one of claims 1 to 10, wherein the protein encoding sequences of the polynucleotide comprise 5' regions which encode: (i) a peptide which is capable of targeting the translation products of the regions to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls; and/or (ii) non- translated translational enhancing sequences. 12. A method according to any one of claims 1 to 1 1 , in which the polynucleotide used to transform the material is modified in that mRNA instability encoding motifs and/or fortuitous splice regions are removed, or plant preferred codons are used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if - in respect of the herbicide resistance conferring regions - the thus modified polynucleotide comprises plant preferred codons, the degree of identity between the protein encoding regions within the modified polynucleotide and like protein encoding regions endogenously contained within the said plant and encoding substantially the same protein is less than about 70%>. 13. A method according to any one of claims 1 to 12, in which the 4-HPPD inhibiting herbicide is selected from the group consisting of isoxaflutole, diketonitriles such as 2-cyano-3-cyclopropyl-l-(2-SO 2 CH 3 -4-CF 3 -phenyl)propane-l,3-dione and 2-cyano- 3-cyclopropyl-l-(2-SO 2 CH 3 -4-2,3Cl 2 phenyl)propane-l ,3-dione, triketones such as sulcotrione, and mesotrione (BSI-proposed), pyrazolynate and pyrazoxyfen. 14. A method according to any one of claims 1 to 13, wherein the herbicide is applied post-germination. 15. A moφhologically normal fertile (or male sterile) whole plant resulting from the method of any one of claims 1 to 14, the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny. - 13 - 16. A plant according to claim 15 selected from the group consisting of banana, cotton, maize, tomato, vines. 17. Use of a polynucleotide comprising a region encoding a phytoene desaturase in the production of plant material which is resistant or tolerant to herbicides which - in vitro - inhibit the enzyme 4-HPPD. 18. A method of selectively controlling weeds in a field, the field comprising weeds and crop plants, the method comprising application to the field of a herbicide which - in vitro - is capable of inhibiting the enzyme 4-HPPD, characterised in that the plants have been transformed with and express the coding regions of a polynucleotide comprising a sequence encoding a phytoene desaturase. 19. A method according to claim 18 wherein the polynucleotide is that mentioned in any one of claims 2 to 12. 20. A method according to either of claims 18 or 19, wherein the said herbicide is selected from the group consisting of, isoxaflutole, diketonitriles such as 2-cyano-3- cyclopropyl-l-(2-SO 2 CH 3 -4-CF 3 -phenyl)propane-l ,3-dione and 2-cyano-3- cyclopropyl-l-(2-SO 2 CH 3 -4-2,3Cl 2 phenyl)propane-l,3-dione, triketones such as sulcotrione, and mesotrione (BSI-proposed), pyrazolynate and pyrazoxyfen. 21. A method according to any one of claims 18 to 20, wherein the field is treated with a pesticide selected from the group consisting of a fungicide, insecticide and nematicide, either prior to or post application to the field of the herbicide.

Inventors: BRYAN IAN BENNETT, SHIPTON CATHERINE ANN, BRYAN, IAN, BENNETT, SHIPTON, CATHERINE, ANN
Applicants: BRYAN IAN BENNETT, SHIPTON CATHERINE ANN, ZENECA LTD, BRYAN, IAN, BENNETT, SHIPTON, CATHERINE, ANN, ZENECA LIMITED
Application year: 1999
Main classification: C12N15
IPC codes: A01H5, C12N9, C12N15