MXPA97009049A - Control of maize root worm through the formation of limonene in transgeni plants - Google Patents

Abstract

The present invention relates to a method for protecting a monocotyledonous plant against infestation by Diabrotica spp. Larvae, which comprises inserting into the plant genome at least one nucleotide sequence coding for limonene cyciase and at least one sequence of nucleotide coding for GPP synthase, each of the sequences in an appropriate reading frame relative to the transcription initiator and promoter sequences active in the plant cause the expression of the enzymes at levels that provide for the production of a quantity of protective plant effective limonene in tissues of the plan

Description

CONTROL OF MAIZE ROOT WORM THROUGH THE FORMATION OF LEMONENE IN TRANSGENIC PLANTS DESCRIPTION OF THE INVENTION TECHNICAL FIELD This invention relates to the proportion of corn and other plants that produce any level or low levels of limonene with resistance to the Southern Corn Rootworm and other insect pests that cause the expression of limonene in plant tissues in sufficient quantities to repel, annihilate or inhibit those pests.

BACKGROUND OF THE INVENTION Numerous insects are serious pests of common agricultural grains. One method of controlling insects has been to apply organic, semi-organic or organometallic insecticidal chemicals to the grains. This method presents numerous environmental problems known in the art and public health. A very recent method to control insect pests has been the use of biological control organisms, which are typically natural predators of problematic insects. These include other insects such as wasps, fungi such as milk spore fungi, and bacteria such as Bacillus thuringiensis cv. , commonly referred to as "Bt". However, it is difficult to apply biological control organisms to large areas, and it is even more difficult to make those living organisms stay in the treated area for an extended period. Even more recently, recombinant DNA techniques have been provided which have given the opportunity to insert into the plant cells cloned genes expressing insecticidal toxins derived from biological control organisms such as Bt. This technology has given rise to additional aspects with respect to the resistance of the final insect to well-known naturally occurring insect toxins, particularly in the heavy selection pressure part, which may occur in some areas. Thus, there is a continuing need to identify naturally occurring insecticidal toxins, which can be formed through plant cells directly by expression of an individual structural gene, which is not normally present in the plant. The Southern Corn Rootworm (Diabotrica undecimpunctata howardi Barber) is a particularly difficult pest to control or eradicate. Attacks the plant below the soil line, where the insecticides have difficulty or can not be effectively applied. In addition, it is resistant to a number of other effective chemical and biological control agents, including Bt toxins and some lecithins. Limonene, 1-methyl-1 - (1-methylethenyl) cyclohexane; p-me nta-1, 8-diene (Entry No. 5371, Merc k I ndex 1 1 th Ed.), exists naturally in several ethereal oils, particularly oils of lemon, orange, alcacavea, abesón and bergamoto. He is a valuable industrial chemist. Some limonene is prepared by extracting plants from the mint families, a large amount is obtained from citrus oils, which are typically 80-90% limonene, and some is obtained from pine oil. It is also synthesized chemically and finds use as a solvent and cleaning agent (in the manufacture of synthetic pine oil), and as an expectorant, as a wetting and dispersing agent, as a monomer in the manufacture of various polymeric resins, as a flavoring and a precursor in the synthesis of the flavoring carvone, and as a storage polymerization inhibitor of the tetrafluoroethylene monomer used in the manufacture of polytetrafluoroethylene (PTFE).

DESCRIPTION OF THE INVENTION It has now been determined that limonene is especially effective in the control of Southern Corn Rootworm. Accordingly, this invention provides a method for killing, repelling or inhibiting larvae of Diabrotica j nd ecimpunctata howardi, which comprises administering larvae to the larva enterally., repellent or inhibitor of limonene feeding. In this description of the invention, the term "annihilate" has its usual meaning and is measured by counting the larvae that die during a measurement period. "Inhibition" is used to represent the inhibition of larval growth, as measured by larval weight and weight gain of larvae that do not die during the measurement period, and "repel" is used to represent no preference for a treated plant in the form of this invention as a food source although growth may not be inhibited and death may not occur. Repellency is measured by comparing numbers of infested larvae treated against control plants. In the practice of this invention, limonene can be effectively applied to plants, harvested plant materials, or derived products consumed by plants by spraying, microencapsulated beads, or other formulation common to the insecticidal technique. Care must be taken that the formulation is such that the limonene does not damage the green tissues of the plant. Alternatively, limonene can be incorporated into the tissues of a susceptible plant, so that during the infestation of the plant, the larva consumes larvicidal amounts of limonene. One method to accomplish this is to incorporate the limonene into a non-phytotoxic vehicle, which is adapted for systemic administration to susceptible plants. This method is commonly employed with insecticide materials that are designed to attack insect chewers and is within the eyes of one skilled in the art of the formulation of insecticides and larvicides. Alternatively, a dietary trap containing limonene may be employed, with, optionally, a pheromonal aggregate or other material attractive to the larva. By "transgenic plant" means any plant or plant cell that has been transformed by the introduction, stable and heritable incorporation, into the subject plant or the plant cell, either of a native DNA that is under the control of a different promoter the promoter that typically drives the expression of that DNA in a wild type plant, and that has been introduced back into its host plant, or foreign DNA, i.e., DNA encoding a protein not normally found within the species of the plant. "Seedling" refers to a plant sufficiently developed to have a stem and a root that reproduce asexually by cell culture. "Explanta" refers to a section or piece of tissue from any part of a plant for cultivation. The term "callus" (cicatricial woody tissue) and its plural "corns", refers to an unorganized group of cells formed in response to cut, separation or other damage that affects the tissue of the plant. The pieces cut from the plant tissue and the isolated cells can be induced to form the callus under the appropriate culture conditions. The callus can be kept in the culture for a considerable time by transferring or subculturing the parts of the callus to a fresh medium at regular intervals. The transfer of the callus to a liquid medium leads to the dispersion of the tissue and the formation of a plant cell suspension culture. The callus can be induced to undergo organized development to form rods and roots. "Embroide" refers to a structure similar in appearance to a plant zygotic embryo. By the term "taxon" in the present one means a unit of botanical classification of genes or lower. In this way, it includes genus, species, crops, variety, variant and other minor taxonomic groups that lack a consistent nomenclature. "Somatic hybrids" and "somatic hybridity" generally refer to the stable combination of cellular materials, being protoplast / cytoplasts or protopl asto / cytoplasts combi nations, and include cybrids and cybridization. A "replica" is any genetic element (for example, plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; that is, capable of replication under its own control. As used herein, the term "nucleotide sequence" means a sequence of AD N or RNA, and may include an AD Nc, genomic DNA, or a synthetic DNA sequence, a structural gene or a fragment thereof. , or a mRNA sequence, which encodes an active or functional polypeptide. A "vector" is a replica, such as a plasmid, phage or cosmid, to which another DNA segment can be attached in order to realize the replication of the attached segment. A "coding sequence" of DNA is a DNA sequence, which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a start codon at the 5'-terminus (amino) and a translation stop codon at the 3'-terminus (carboxy). A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3 'to the coding sequence. A "promoter sequence" or a "promoter" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating the transcription of a downstream coding sequence (3 'direction). For the purpose of defining the present invention, the promoter sequence is joined at its 3 'terminus by the translation initiation codon (ATG) of a coding sequence and extended upstream (5' direction) to include the minimum number of bases or elements necessary to start the transcription. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for RNA polymerase binding. Eukaryotic promoters will usually, but not always, contain "TATA" boxes and "CAT" boxes.

"DNA control sequences" collectively refers to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. A coding sequence is "operably linked to" or "under the control of" control sequences in a cell when the RNA polymerase binds to the promoter sequence and transcribes the mRNA, which is then translated into the polypeptide encoded by the coding sequence. A "host cell" is a cell that has been transformed, or is capable of undergoing transformation, by an exogenous DNA sequence. A cell has been "transformed" by endogenous or exogenous DNA when such DNA has been introduced into the cell membrane. DNA can or can not be integrated into chromosomal DNA (covalently linked) by making the genome of the transformed cell. In prokaryotes and yeasts, for example, the DNA can be maintained in an episomal element, such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which DNA has been integrated into the chromosome, so that it is inherited by sister cells through chromosome replication. This stability is demonstrated through the ability of the eukaryotic cell to establish cell lines or clones composed of a population of sister cells containing the DNA. A "clone" is a population of cells derived from a ndividual cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. Two DNA, RNA or polypeptide sequences are "substantially homologous" when at least about 85% (preferably about 90%, and most preferably at least about 95%) of the nucleotides or amino acids coincide over a length defined from the molecule. AD N sequences that with substantially homologs can be identified in a low Southern hybridization experiment, e.g., severe conditions, as defined by that particular system. The definition of appropriate hybridization conditions is within the technical experience. See, for example, Maniatis et al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1982; I I Brown, T. A. , Gene Cloning: An Introduction (2nd of.) Chapman & Hall, London (1990). A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not in association with the other molecule by nature. In this way, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacterium. Another example of a heterologous coding sequence is a construct, wherein the coding sequence by itself is not found in nature (eg, synthetic sequences having different codons from the native gene). "Heterologous" DNA also refers to DNA not found within the host cell by nature. The allelic variation or mutational cases of natural existence do not give rise to a heterologous region of DNA, since as are these terms used in the present. "Native", "autologous" or "endogenous" DNA, as used herein, refers to DNA that is typically present in the host by nature. The term "polypeptide" as used herein, is used in its broadest sense, ie, any amino acid polymer (dipeptide or higher) linked through peptide bonds. In this way, the term "polypeptide" includes proteins, oligopeptides, protein fragments, analogs, mutefins, fusion proteins and the like. The term also encompasses amino acid polymers as described above and include additional portions without amino acid. Thus, the term "polypeptide" includes glycoproteins, lipoproteins, phosphoproteins, metalloproteins, nucleoproteins, as well as other conjugated proteins. The term "polypeptide" contemplates polypeptides as defined above that are recombinantly produced, isolated from an appropriate source, or synthesized. In view of the ability to transform grain plants to express various heterologous compounds, it may be desirable to transform the corn plants to express limonene, so that by consuming the plant tissues, the larvae could also consume toxic or inhibitory amounts of limonene. However, since most gene products are peptides, limonene is not a peptide or a peptide derivative and is not expressed from genes in the form of a peptide or a peptide derivative and is not expressed from genes in the form of a peptide or peptide derivative, but is produced enzymatically as a secondary metabolite within the cells of some plants. It has now also been found that the biosynthetic apparatus necessary for the production of limonene can not be present in many plant cells, which do not produce limonene, or can not produce detectable, insecticidally effective amounts of limonene, and this seems to include the cells of corn. Said plant cells must be engineered with at least one enzyme that can be produced through the expression of exogenous (heterologous) genes. One of these enzymes is limonene synthase, also known as limonene cyclase, which can be directly synthesized from limonene of geranyl piprofosphate (GPP), which is widely found in both prokaryotic and eukaryotic cells, although, as will be discussed , in some cases it is not produced in sufficient quantities to be insecticidally effective amounts of limonene. Since the genes encoding limonene pair synthase can be synthesized, either directly using a DNA sequence obtained by working from the known amino acid sequence of the limonene synthase and preferably using preferred codons for plants, or by naturally cloning the limonene , the resulting sequence can be inserted into an appropriate expression cassette, and introduced into the cells of a susceptible plant species or a suitable endophytic bacterium, so that an especially preferred embodiment of this method involves insertion into the plant genome or bacterium of a DNA sequence encoding the limonene synthase, in an appropriate reading frame in relation to the transcription initiator and promoter sequences in the plant or plant or bacterium. The transcription and translation of DNA under the control of regulatory sequences can, in some cases, cause the expression of the enzyme at levels that provide an insecticidal amount of limonene in the tissues of the plant, which are normally infested by the larvae. As an illustration, it can be seen that Colby et al., In Keystone Symposium on Crop Improvement via Biotechnology: An International Perspective, Keystone, Colorado, USA, April 10-16, 1992, as reported in J. Cell Biochem. Suppl. 16, F, 230 (1992) have isolated and characterized cDNA encoding spearmint limonene cyclase. To isolate and study the genes encoding limonene synthase (sic) and to produce enough for structural studies, they used normal methods to extract RNA from young leaves of Mentha spicata and constructed to a cDNA library in lambda ZAP XR (Stratagene) from poly (A) + RNA. They designed three degeneration oligonucleotides based on the internal amino acid sequences obtained from the Edman degradation of purified limonene synthase and were classified 250, 000 clones to identify six positive clones that can hybridize the three oligonucleotides. The resulting clones can be used in the methods of this invention, which involve transformation of plants. However, Colby and others do not indicate any appreciation of the value of the enzyme in the worm resistance conference to the plants. In addition, it has been discovered that in certain plants, including corn, at least one additional gene, encoding GPP synthase, is required for the generation of plants resistant to the rootworm. Due to the fact that natural levels of GPP are low in such plants, inadequate amounts of GPP may exist for the production of limonene in maize or other plants that are transformed only with the limonene synthase gene. This is in contrast to GP levels in other plant species, such as spearmint, which are much more superior. In such species, some GPP is used to generate limonene, something to generate other metabolites. Since GPP is derived from a path that is common among plant species, the introduction of both the GPP synthase gene and the limonene synthase gene in the plant species such as corn, can produce transgenic plants capable of expressing GPP, and then the limonene of metabolic product, at levels sufficient to confer resistance to insect pests, particularly the corn rootworm. The plant, which can be benefited by this invention, is preferably a plant susceptible to infestation and damage by the larvae of Diabrotica undecimpunctata howardi or whose harvested material is subjected to attack by larvae of that insect. A prime example is corn (Zea mays). The transformation of maize cells, and the regeneration of transformed cells to produce a fertile, complete, transformant offspring (RO) has also been reported by several centers and is now routine. However, this should not be construed as limiting, since this species in the past has been among the most difficult commercial grains to reliably transform and regenerate, and these insects (under other common names) also infest certain grains. In this manner, the methods of this invention are readily applicable and conventional techniques to numerous plant species, if they are susceptible to Diabrotica undecimpunctata howardi, including, without limitation, species of the genus Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella. , Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Synapse, Atropa, Capsicum, Datura, Hyoscyamus, Licopersicon, Nicotiana, Solarum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus , Antirrhinum, Hemerocallis, Nemesia, Pelagonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lotium, Triticum and Datura. Preferred plants to be transformed according to the methods of this invention are cereal grains, including corn, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean, grains. of fiber, such as cotton, fruit grains, such as melons and vegetable grains including onion, pepper, tomato, cucumber, pumpkin, carrot, cruciferous (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce. The DNA sequence or sequences, which when expressed impart insecticidal activity, are either a structure gene coding for limonene synthase, or a combination of that structural gene and a structural gene encoding GPP synthase, depending on the level of GPP produced in the wild type plant. It has been found that limonene has sufficient insecticidal activity (larvicide) to operate when formed as the final product of a plant cell expression system. That is, since certain compounds have some larvicidal activity at high concentrations in IK form pure, the expression of plant cell at such high concentrations is either not possible in a living plant cell system, or it is not reliable if the commercially useful characteristics of the plant are to be conserved in terms of production of oils, starches , fibers or other materials. Limonene, on the other hand, is not directly expressed as the gene product, and the peptide or peptides, which are expressed in the methods of this invention, are an enzyme that can catalyze the synthesis of large amounts of limonene for accumulation in the tissues of the transformed plant (limonene synthase), and in cases where required, an enzyme that can catalyze the synthesis of large quantities of the substrate for limonene synthase. A specific promoter in the tissue (or promoters) can be used in any case, where it may be desirable to localize the production of limonene to an infested site or to a tissue which is efficient in the production of the enzyme. Since the Southern corn rootworm tacks the roots, an especially preferred tissue specific promoter is a root specific promoter. To carry out this invention, it will be appreciated that numerous cassettes and plant expression vectors are known in the art. By the term "expression cassettes" represents a complete set of control sequences including initiation, promoter and termination sequences, which function in a plant cell, when flanking a structural gene in the appropriate reading frame. The cassettes of frequent expression and preferably contain a classification of restriction sites suitable for the insertion excision of any desired structural gene. It is important that the cloned gene has a start codon in the correct reading frame for the structural sequence. In addition, the plant expression cassette preferably includes a strong promoter sequence at the end, to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the RNA delivery courier. An example of said preferred "blank" expression cassette in which the DNA sequence of the present invention can be inserted, is the plasmid pPH1414 developed by Beach et al. of Pionner Hi-Bred I nternational, I nc. , Johnston, IA. Highly preferred plant expression cassettes will be designed to include one or more selectable marker genes, such as herbicide tolerance genes, or kanamycin resistance genes. By the term "vector", herein is meant a DNA sequence that is capable of replicating and expressing a foreign gene in a host cell. Typically, the vector has one or more endo-nuclease recognition sites, which can be cut in a predictable manner through the use of appropriate enzyme. Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic resistance or herbicide, which then serve as selectable markers to identify and separate transformed cells. Preferred screening agents include kanamycin, chlorosulfuron, phosphonothricin, hygromycin and methotrexate, and preferred markers are genes that confer resistance to these compounds. A cell in which the foreign genetic material is a vector, is functionally expressed and has been "transformed" by the vector and is referred to as a "transformant". A particularly preferred vector is a plasmid, by which is meant a DNA molecule of circular double structure that is not a part of the chromosomes of the cell. As mentioned above, the genomic, synthetic cDNA encoding the limonene synthase enzyme, and as a genomic, synthetic and cDNA necessary encoding the GPP synthase enzyme, can be used in this invention. The vector of interest can also be partially constructed from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone, or a combination thereof. When a sequence gene or sequences are / are at hand, genetic constructs are made, which contain the regulatory sequences necessary to provide efficient expression of the gene in the host cell. According to this invention, the genetic construct will contain (a) at least one genetic sequence encoding the enzyme responsible for making the larvicide compound of interest and (b) one or more regulatory sequences operably linked on both sides of the gene or genes structural Typically, the regulatory sequences will be selected from the group comprising promoters and terminators. Regulatory sequences can be from autologous or heterologous sources. In preferred embodiments involving the use of both the limonene synthase gene and the GPP synthase gene, each gene will be under the control of its own regulatory sequences. Promoters that can be used in the genetic sequence include, us, oes, phaseolin, CaMV, FMV, ubiquitin, and other promoters isolated from plant DNA or other sources, both natural and synthetic. An efficient plant promoter that can be used is an overproduction plant promoter. The overproduction plant promoters that can be used in this invention include the promoter of the small subunit (ss) of soy ribulose 1,5-bisphosphate carboxylase (Berry-Lowe et al, J. Molecular and App. ., 1: 483-498 (1982)), and the promoter of the chlorophyll ab binding protein. However, these two promoters are known to be induced by light in cells of eukaryotic plants (see, for example, Genetic Engineering of Plants. An Agriculture! Perspective, Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al. , J. Biol. Chem. 258: 1399 (1983), and P. Gunsmuir, et al. , J. Molecular and App. Getb., 2: 285 (1983)) and may be less desirable when root expression is desired. An especially preferred constitutive promoter is the 35S promoter of Cauliflower Mosaic Virus. Root-specific promoters are especially preferred for control of Southern corn rootworm while minimizing the production of limonene in the agronomically valuable parts of the plant, such promoters are also known and can be selected from many available from the technique and isolated from new compatible species. For example, Hirel, B., Marsolier, M.C. , Hoarau, A., Hoarau, J., Brangeon, J., Schafer, R., and Verma, D.P.S. , Plant Molecular Biology. October 1992. v. 20 (2) p. 207-218, describe a specific glutamine synthase gene in the soybean root. Keller, B. and Baumgartner, C, The Plant Cell. October 1991. v.3 (10) p. 1051-1061, describe a specific control element at the root in the GRP gene 1.8 of French bean. Sanger, M., Daubert, S., and Goodman, R.M., Plant Molecular Biology March 1990. v. 14 (3) p. 433-443, discuss the specific promoter in the root of the Manipin Synthase (MAS) gene of Agrobacterium tumefaciens. Miao, G.H., Hirel, B., Marsolier, M.C., Ridge, R.W., and Verma, D.P.S., The Plant Cell. January 1991. v. 3 (1) p. 11-22, describes a full-length cDNA clone encoding cytosolic flutamine synthase (GS), which is expressed in roots and nodules of the soybean root. Bogusz, D., Llewellyn, D.J., Craig, S., Dennis, E.S., Appleby, C.A., and Peacock, W.J., The Plant Cell. July 1990. v. 2 (7) p. 633-641, discuss two specific root promoters isolated from hemoglobin genes of Parasponia andersonii without fixed nitrogen legume and Trema tomentosa without legume fixation without related nitrogen. The promoters of these genes were linked to a beta-glucuronidase reporter gene and introduced both in the non-leguminous Nicotiana tabacum and in the Lotus corniculatus legume, and in both cases specific promoter activity was observed in the root. Leach, F. and Aoyagi, K., Plant Science (Limerick) 1991, 79 (1): 69-76, describe their analysis of the promoters of the highly expressed root induction genes rolC and rolD of Agrobacterium rhizogenes. They concluded that the enhancer and specific DNA determinants in the tissue are disassociated in those promoters. Teeri, T. H., Lehvaslaiho, H., Franck, M., Uotila, J., Heino, P., Palva, E. T., Montagu, M. van, and Herrera-Estrella, L., EM BO Journal. 1989, 8 (2): 343-350, using gene fusions to lacZ to show that the Agrocaterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the tip of the root and that the ten TR2 ' it was specific at the root in the intact plant and was stimulated by the formation of wounds in the leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene. The TR1 'gene fused to NPTI I, (in neomycin phosphotransferase I I) showed similar characteristics. The expression cassette comprising the structural gene or genes of the method of the invention, operably linked to the desired control sequences can be ligated to a suitable cloning vector. In general, the plasmid or viral vectors (bacteriophage) containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector typically carries an origin of replication, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes that confer resistance to selected antibiotics or herbicides are used. After the genetic material is introduced into the target cells, cells and / or colonies successfully transformed from cells can be isolated by selection based on these markers. Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant amounts for introduction to the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and S. marcescens. Eukaryotic hosts such as yeast and filamentous fungi can also be used in this invention. The isolated cloning vector will then be introduced to the plant cell using any convenient technique, including electroporation (in protoplasts), retroviruses, microparticle bombardment, and microinjection, to the cells of monocotyledonous or dicotyledonous plants, in a cell or culture culture. tissue, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence or expression cassette sequences of the plant. Preferably, the moncotyledonous species will be selected from corn, sorghum, wheat and rice, and the dicotyledonous species will be selected from soybean, sunflower, cotton, rapeseed (either edible or industrial), alfalfa, tobacco and Solanaceae such as potato and tomato. . Using known techniques, protoplasts can be regenerated and the cell or tissue culture can be regenerated to form fully fertile plants that carry and express the desired gene for the selected protein. Accordingly, a highly preferred embodiment of the present invention is a transformed corn plant, the cells of which contain as foreign DNA in at least one copy of the DNA sequence or sequences of an expression cassette, which directs the expression of limonene synthase, and in some cases also the GPP synthase. This invention also provides methods for imparting resistance to Diabrotica undecimpunctata howardi to plants of a susceptible taxon, comprising the steps of: a) culturing cells or tissues from at least one plant of the taxon, b) introducing cells into the cell culture or of tissue at least one copy of an expression cassette comprising a structural gene encoding either limonene parasynthase or both for limonene synthase and for GPP synthase, operably linked to plant regulatory sequences, which cause expression of the enzyme in the cells, and c) regenerating whole plants resistant to the insects of the cell or tissue culture. Once the whole plants have been obtained, they can be sexually or clonally reproduced in such a way that at least one copy of the sequence provided by the expression cassette is present in the progeny cells of the reproduction. Alternatively, once the individual transformed plant has been obtained by the above recombinant DNA method, conventional plant breeding methods can be used to transfer the gene or structural genes and associated regulatory sequences through interlacing or retro-interlacing. Such intermediary methods will comprise the additional steps of a) sexually crossing the insect resistant plant with a taxon plant susceptible to the insect.; 2H b) recover the reproductive material of the crossbreeding progeny; and c) develop plants resistant to the insect of the reproductive material. When desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially conserved by expanding this method to include the additional steps of repetitively: a) backcrossing the progeny resistance to the insect with plants susceptible to the insect of the susceptible taxon; and b) selecting for expression of insect resistance (or an associated marker gene) or limonene production among the backcross progeny, until the desired percentage of the characteristics of the susceptible taxon is present in the progeny together with the gene that imparts the resistance to insects. The term "taxon", in the present, means a genus botanical classification unit or lower. In this way, it includes genus, species, crops, varieties, variants and other minor taxon groups that lack a consistent nomenclature. It will also be appreciated by those skilled in the art that the plant vectors provided herein may be incorporated into Agrobacterium tumefaciens, which may then be used to transfer the 2 < vector to cells of susceptible plant, mainly of dicotyledonous species. Thus, this invention provides a method for imparting insect resistance in dicotyledonous plants susceptible to Agrobacterium tumefaciens, wherein the expression cassette is introduced into the cells by infection of the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include a plant expression cassette expressing limonene synthase in the form of this invention. Finally, insect pests of harvested material, including stored grain, may also be targets for the methods of this invention. In view of this, the invention also provides methods for annihilating Diabrotica undecimpunctata howardi larvae in harvested materials and products obtained from harvested materials, comprising the application of limonene to the grain or causing either the limonene synthase or the GPP synthase and the Limonene synthase are expressed in the grain. The following description further illustrates the compositions of this invention and the methods for making and using them. However, it will be understood that other methods, known to those skilled in the art which may be equivalent, may also be employed.

EXAMPLES 1-6 Inhibition of insect larvae and toxicity tests for Worm of Southern Corn and European Corn Weevil (ECB). Bioassay diets were prepared as described by Czapla and Lang in "Effect of Plant Lectins on the Larval Development of European Corn Borer (Lepidoptera: Pyralidae) and Southern Corn Rootworm (Coleptera: Chrysomelidae) ", J. Econ. Entomol , Environment! .. 83: 2480-85 (1990), except that the low melting temperature agarose replaced the regular agarose so that the diets could be cooled to 37 ° C before the addition of limonene (an assay used to the regular agarose diet). The results were as follows. In Examples 1-4, the test larvae were Southern Corn Rootworms. In Examples 5-6, the test larvae were European Corn Weevil. The results of each experiment represent the average of 16-32 insects. All limonene concentrations (ppm) are by weight. The SCR data indicate that limonene is effective against larvae, but when limonene was used in the same protocol against ECB, little or no effect was observed as indicating that limonene is not effective against some relatively susceptible grain pests, common. (control mortality treated | Mortality correlation% = 100 x 100-control control weight - treated weight% reduction in weight = 100 x control weight Example: 1 2 limonene% Cor. % weight% Cor. % wt wt. mortal wt. mortal reduction reduction ,000 57 64 80 8b 1,000 49 52 19 30 100 57 52 15 6 3 4 Example: limonene% Cor. % weight% Cor. % wt wt. wt. mortal wt. reduction wt. reduction 96 64 44 0 7 0 22 0 26 0 19 0 Results of SCR Promedie y. Limonene% Cor. % weight ppm Mortal Reduction 10,000 69 54 1,000 24 21 100 29 15 Example: 5 6 limonene% Cor. % weight% Cor. % weight pp Fatal Deadly Reduction Reduction ,000 0 28 0 0 1,000 0 16 0 0 100 0 1 0 7 Average ECB results: limonene% Cor. % weight ppm Mortal Reduction 10,000 0 14 1,000 0 8 100 0 4 EXAMPLE 7 Corn callus cultures were transformed by bombardment of microprojectiles using plasmids containing a gene cloned modified for the enzyme of timonene synthase (limonene silasa) activated by a ubiquitin promoter and a ubiquitin intron and followed downstream by a PI N terminator. -II. Complete fertile plants of the transformed callus were regenerated and analyzed for limonene and limonene synthase. The representative results of a series were as follows Plant Callus # clone Callo LS1 limoneno2 Plant Plant LS Limonene C6 22 539 ND 3 1950 ND 7 2000 ND C19 9900 ND 6 2100 ND C3 2550 ND 3 1900 ND 4 1 650 ND 1LS = Limonene Synthase 2ND = not detected above 0.5 nq / g freshly prepared by weight.

In other experiments, transgenic maize plants and tissues were produced that exhibited high expression of the limonene synthase protein. The enzyme was extracted from transformed plants and tissues and allowed to react with tritium-labeled geranyl pyrophosphate (GPP). The extracted enzyme converted GPP to limonene, showing that a functional enzyme was produced. In addition, western stains were performed and confirmed the presence of the LS protein in transgenic tissues, but not in negative control tissues. The seed was collected from the transformed plants. In summary, the complete fertile transformed plants that have been produced express active limonene cyclase (synthase) according to the teaching of the previously identified application.

EJ EM PLO 8 In view of the stated results, in Example 7, the demonstration of active limonene synthase expression in transformed, fertile, complete plants, larvantly effective amounts of limonene can be produced in such transgenic plants where sufficient substrate is present for the enzyme of limonene synthase to act on them. Therefore, corn callus cultures can be transformed by art-recognized microprojectile bombardment methods, using plasmids containing genes encoding both the limonene synthase enzyme and the GPP synthase enzyme, activated, for example, through one or more promoters (a ubiquitin promoter, for example) and followed downstream by, for example, a PI NI I terminator. Fertile, complete plants of the transformed callus were regenerated and analyzed for the presence of and / or activity of both enzymes, and also analyzed for the presence of limonene. Alternatively, the callus can be generated from transgenic plants that contain and express the limonene synthase transgene, and said callus cultures can be transformed as described above, except that such calluses can be transformed using plasmids containing a gene encoding the GPP synthase protein. Transgenic, fertile, regenerated whole plants of such transformed calli can be expected to produce larvicidally effective amounts of limonene.

Claims (3)

R E I VI N D I C A C I O N S

1. A method for protecting a monocot plant against infestation by Diabrotica spp. Larvae, which comprises inserting into the plant genome at least one nucleotide sequence coding for limonene cyciase and at least one nucleotide sequence coding for nucleotide synthase. GPP, each of the sequences in an appropriate reading frame relative to the transcription initiator and promoter sequences active in the plant cause the expression of the enzymes at levels that provide for the production of an effective amount of protective plant of limonene in tissues of plant.

2. The method according to claim 1, characterized in that the monocotyledonous plant is a maize plant (Zea mays L).

3. The method according to claim 1 or 2, characterized in that one or both promoters are a specific promoter in the tissue. The method, according to claim 3, further characterized in that one or both promoters are specific promoters in the root. SUMMARY Limonene is effective in controlling Southern corn rootworm. This can be applied as a conventional insecticide, or the plants can be transformed with a gene that codes for an enzyme that synthesizes limonene from a substrate available in the plant. The transformed plants are resistant to infestation by Southern corn rootworm.