MXPA99005746A - Methods for conferring insect resistance to a monocot using a peroxidase coding sequence - Google Patents

Abstract

The present invention relates, in general, to methods and compositions for controlling insects in monocotyledonous plants (monocots), particularly maize. More precisely, the present invention relates to (1) a method for controlling insects comprising feeding or contacting an insect with an insecticidal amount of transgenic monocotyledonous plant cells comprising a recombiant DNA sequence comprising a coding sequence encoding peroxidase and (2) a fertile transgenic monocot plant comprising a recombinant DNA sequence comprising a coding sequence encoding peroxidase.

Description

METHODS FOR CONFERING INSECT RESISTANCE TO AN ONOCO-TILEDONE BY USING A PEROXIDASE CODING SEQUENCE The present invention relates generally to methods and compositions for controlling insects in monocotyledonous plants, particularly corn. More precisely, the present invention relates to: (1) a method for controlling insects, which comprises feeding or contacting an insect with an insecticidal amount of transgenic monocotyledonous plant cells comprising recombinant DNA comprising a encoding sequence encoding peroxidase, and (2) a transgenic monocotyledonous transgenic plant comprising recombinant DNA comprising a coding sequence encoding peroxidase. Insect pests are the main factor in the loss of commercially important agricultural crops in the world. Wide-spectrum chemical pesticides have been widely used to control or eradicate pests of agricultural importance. Although insecticides have been effective in controlling most of the harmful insects, there are considerable problems associated with the use of these compounds. Insecticides are expensive and expensive to apply. Repeated applications are often needed for effective control. There is also a concern that insects have or will have resistance to many of the chemicals used in their control. Insecticides often annihilate beneficial insects that are pollinators or that feed on herbivorous insects. Additionally, there are environmental risks associated with the long-term use of chemical insecticides. Pest management programs are being introduced that reduce the use of chemical insecticides. These programs include the improvement of crops through selection, the use of biological control agents and insect predators, and the incorporation of insect-resistant genes through breeding programs and genetic engineering. The genes most widely used for genetic engineering are the crystal protein genes from Bacillus thuringiensis. See, for example, Rice et al., European Patent Number EP-A-292,435, and Koziel et al., International Publication Number WO 93/07278. Most of the crystal proteins made by Bacillus are toxic to the larvae of the insects of the orders Lepidoptera, Diptera, and Coleoptera. In general, when a susceptible insect ingests an insecticidal crystal protein, the crystal is solubilized, and acts as a toxic fraction. To prevent the development of insects that are resistant to these toxins, additional toxins are needed that have additive or synergistic effects. Peroxidases are a subclass of oxide-reductases that use peroxide such as H202 as a substrate. Peroxidases are monomeric glycoproteins containing heme capable of binding to the divalent cations (mainly Ca2 +, but also Mn2 +) (Maranon and Van Huystee, Phytochemistry 37: 1217-1225 (1994)). The prosthetic groups for peroxidase have different roles. Although the heme group is involved in catalysis, divalent cations stabilize the heme fraction, and glycosyl groups can help stabilize peroxidase by decreasing its rate of change (Maranon and Van Huystee, Phytochemistry 37: 1217-1225 (1994)) . Peroxidases are often grouped into anionic, cationic, and neutral forms according to their migration on isoelectric focusing gels. Although it is considered that as enzymes they have a broad substrate specificity, they appear to have some substrate "preferences" for different isoenzymes (Van Huystee, Ann. Rev. Plant Physiol., 205-219 (1987)). There are several types of peroxidases and related enzymes, including guaiacol peroxidase, NADH peroxidase, cytochrome-C peroxidase, catalase, glutathione peroxidase, L-ascorbate peroxidase, and manganese peroxidase. In plants, peroxidases are monomeric proteins that are highly complex enzymes whose activities are tightly regulated by the plant. Peroxidases are critical in the biosynthesis of cell walls of the plant. Peroxidases promote the peroxidative polymerization of monolignoles coniferyl, rcoumaril, and sinapyl alcohol in lignin (Greisbach, In: The Biochemistry of Plants Ed. Conn, Academic, New York, pages 457-480 (1991)). Different plant species have different proportions of the monolignol species assembled in a semi-random form (Hwang et al., Carbohydrate Polymers 12: 11-88 (1991)). Lignification serves to strengthen and strengthen cell walls. The overall result is a hardening of the plant tissue. An anionic tobacco peroxidase was used to transform N. tabacum and N. sylvestris (Lagrimini, Plant Cell 2: 1-18 (1990)); Lagrimini, Plant Physiology 96: 511-583 (1991)). These transgenic plants on constitutively expressed an anionic tobacco peroxidase from a 35S promoter. The most striking phenotype of peroxidase overexpression was a chronic wilt that begins at about the time of flowering. In addition, the growth of the plants was retarded, they had smaller compact cells, and they were quickly placed chestnuts in response to the wounds. The same construction was also used to transform tomato plants (Lagrimini et al, J "Am. Soc. Hort. Sci. 117: 1012-1016 (1992)); Lagrimini et al., Hortscience 28: 218-221 (1993) It was also found that these plants wilt severely after flowering, and showed excessive chestnut color and reduced fruit size, and initial studies have shown that some tissues of transgenic tobacco and tomato plants that express a peroxidase gene anionic tobacco, were resistant to some insects (Dowd et al, presentation at the National Meeting of the Entomological Society of America, Indianapolis, December 1993) .Tobacco and tomato are closely related dicotyledons belonging to the same family, the Solanaceae. In contrast, the transgenic monocotyledons of the present invention have a very different physiology, biochemistry, anatomy, and metabolism when stop with the dicotyledons. For example, monocotyledons have different use of codons, use a metabolism of C4 instead of C3, have different fatty acid content, imperfect flowers, and the like. Therefore, it was not known if there would be substrates in the monocotyledons that could be used by peroxidase to control insects. In addition, peroxidases are glycoproteins that must undergo a specific modification after transcription, and incorporation of groups containing heme, to be stable and enzymatically active. Peroxidases are involved in the synthesis of secondary metabolites and lignins whose nature depends on the substrates available in the specific plant. Accordingly, the final products obtained by the expression of the peroxidases may differ from plant to plant. Additionally, resistance to corn cob worms is negatively correlated with the chestnut color of silk, indicating that an increase in peroxidase would lower the resistance (Byrne et al Environ, Entomol., 18: 356-360 (1989)). This eliminates the use of peroxidase to control insects in monocots. In addition, the production of altered lignin in maize (in the jbm mutants) causes a greater susceptibility to insects (Barriere and Argillier, Agronomie 13: 865-816 (1993)). Therefore, it would not be expected that a foreign peroxidase that alters the lignification decreases susceptibility to insects. Furthermore, it was unexpected, from the teachings of Bergvinson et al., The Canadian Entomologist 127: 111-122, 1995, that insect resistance is imparted to plants by hardening the tissues due to peroxidase activity in the former. growth stages. Accordingly, prior to the present invention, the effect of expressing a recombinant peroxidase in monocots was unpredictable. Methods for controlling insects, and insect-resistant monocotyledonous plants are provided. More specifically, the invention provides a method for controlling insects, which comprises feeding or contacting an insect with an insecticidal amount of cells of transgenic monocotyledonous plants comprising recombinant DNA comprising a coding sequence encoding peroxidase, in where the expression of peroxidase confers resistance to insects in the cells of transgenic monocotyledonous plants. The invention also provides a fertile transgenic monocot plant, at least a portion of which comprises cells with recombinant DNA comprising a coding sequence encoding peroxidase, wherein peroxidase expression confers on the monocotyledonous plant a phenotypic trait. The invention also provides a cell, tissue, or seed of a transgenic plant, obtained from the plant described above. The invention also provides transgenic descendants of the plant described above. The invention also provides a cell, tissue, or seed of a transgenic plant, obtained from the offspring described above. The additional objects and advantages of the present invention will become clear from the following description.

FIGURE 1. The plasmid, pJS20293, is shown to contain anionic tobacco peroxidase inserted between (1) the 35S promoter of CaMV linked to the shrunk intron, and (2) the 35S terminator of CaMV. FIGURE 2. Plasmid pUBIAc is shown.

The following definitions will help to understand the present invention. Plant cell: The structural and physiological unit of plants, consisting of a protoplast and the cell wall. The term "plant cell" refers to any cell that is part of, or derives from, a plant. Some examples of cells include differentiated cells that are part of a living plant; Differentiated cells in culture; undifferentiated cells in culture; cells of undifferentiated tissue, such as callus or tumors; Differentiated cells of seeds, embryos, propagules, and pollen. Plant tissue: A group of plant cells organized into a structural and functional unit. Any tissue of a plant in the plant or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue, as mentioned above or otherwise encompassed by this definition, is not intended to be exclusive of any other type of plant tissue. Protoplast: A plant cell without a cell wall. Descending plant: A sexually or asexually derived future generation plant that includes, but is not limited to, progeny plants. Transgenic plant: A plant that has recombinant DNA stably incorporated in its genome. Recombinant DNA: Any DNA molecule formed by joining segments of DNA from different sources and produced using recombinant DNA technology. Recombinant DNA technology: Technology that produces recombinant DNA in vi tro, and transfers the recombinant DNA to cells, where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, the transfer of DNA to protoplasts or cells in different forms, including, for example: (1) naked DNA in circular, linear, or supercoiled forms, (2) DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules, (4) DNA encased in liposomes, spheroplasts, cells or protoplasts, or (5) DNA transferred from organisms other than the host organism (eg, Agrobacterium tumefiaciens).

These and other different methods for introducing the recombinant DNA into the cells are known in the art, and can be used to produce the transgenic cells or the transgenic plants of the present invention. Recombinant DNA technology also includes the methods of homologous recombination described in Treco et al., International Publication Number WO 95/12650 and Treco et al., International Publication Number WO 95/31560, which can be applied to increase peroxidase activity in a monocot. Specifically, regulatory regions (e.g., promoters) can be introduced into the plant genome to increase the expression of endogenous peroxidase. Also included as recombinant DNA technology is the insertion of a peroxidase-coding sequence that lacks selected expression signals in a monocotyledon, and assay of the transgenic monocotyledonous plant for increased expression of peroxidase, due to endogenous control sequences in the monocotyledon . This would result in an increase in the number of copies of the peroxidase coding sequences inside the plant. The initial insertion of the recombinant DNA into the genome of the R ° plant is not defined as performed by traditional plant breeding methods, but instead is by technical methods as described herein.

Following the initial insertion, transgenic offspring can be propagated using essentially traditional breeding methods. Chimeric gene: A DNA molecule that contains at least two heterologous parts, for example the parts derived from previously existing DNA sequences that are not associated in their previously existing states, these sequences having been preferably generated using recombinant DNA technology. Catete expression: A DNA molecule comprising a promoter and a terminator between which a coding sequence can be inserted. Coding sequence: A DNA molecule that, when transcribed and translated, results in the formation of a polypeptide or a protein. Gene: A separate chromosomal region comprising a regulatory DNA sequence responsible for the control of expression, i.e., transcription and translation, and a coding sequence that is transcribed and translated to a different polypeptide or protein. Phenotypic trait: An observable property resulting from the expression of one or more genes.

The present invention relates to methods for controlling insects such as Coleoptera, Diptera, Hymenoptera, Lepidoptera, Malophagous, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermápteros, Isoptera, Anoplura, Siphonaptera, and Trichoptera. Particular examples of these insect pests are European corn borer, corn stem borer, black cut worm, corn cob worm, autumn worm, southwestern corn borer, minor corn borer, borer sugarcane, western corn rootworm, northern corn rootworm, southern corn rootworm, wireworms, northern masked bumblebee, southern masked bumblebee, Japanese beetle, corn beetle, picador corn, corn leaf aphid, corn root aphid, chinch, red-legged grasshopper, migratory grasshopper, corn seed moth, corn-spotted leaf mincer, grass thrush, thief ant, and spider mite two spots. The present invention encompasses any method wherein the expression of peroxidase confers insect resistance in cells of monocotyledonous plants. In a preferred embodiment, the present invention relates to a method for controlling insects by feeding or contacting an insect with an insecticidal amount of cells from transgenic monocotyledonous plants, wherein the genome of these plant cells encodes an enzyme with active of peroxidase. After expression, peroxidase confers insect resistance to the cells of transgenic plants. The transgene encoding peroxidase constitutes an additional gene inserted into the genome of a progenitor plant that does not naturally encode this peroxidase. In a further preferred embodiment, the homologous recombination methods described in Treco et al., International Publication Number WO 94/12650, and Treco et al., International Publication Number WO 95/31560, are used to increase peroxidase activity in a monocot, and therefore, to provide resistance to insects. In a specific manner, regulatory regions (eg, promoters) are introduced into the plant genome to increase the expression of endogenous peroxidase, which increases plant insect resistance. In another preferred embodiment, the present invention relates to the insertion of a peroxidase coding sequence that lacks selected expression signals, in a monocotyledon, and testing the transgenic monocot plant for increased expression of peroxidase due to endogenous control sequences in the monocot. This results in an increase in the number of copies of the peroxidase coding sequences inside the plant. In an additional preferred embodiment, the present invention relates to a method for increasing the copy number of the endogenous peroxidase gene, wherein insect resistance is conferred to the monocotyledonous plant. This method of preference is done using traditional plant breeding methods, or using woven culture techniques. Insect-resistant plants comprise a higher resistance to insects than those found in native, unmanipulated plants, due to higher levels of peroxidase. The invention further relates to a commercial pouch comprising seeds of a monocotyledonous plant transformed with recombinant DNA comprising a coding sequence encoding peroxidase, wherein the expression of peroxidase confers on the plant a phenotypic trait. Within this invention, a commercial bag comprising seeds of a transgenic plant is preferred, wherein peroxidase expression confers on the plant insect resistance or standing possibility. A further preferred object of the invention is this commercial bag, along with label instructions for the use of the seeds contained therein.

Insect Resistance Preferably, the transgenic monocotyledons of the present invention are resistant to insects selected from orders that include, but are not limited to, Coleoptera, Diptera, Hymenoptera, Lepidoptera, Malophagous, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermatators, Isoptera, particularly Coleoptera and Lepidoptera. For the purposes of the present invention, it is recognized that the transgenic plants of the invention can be resistant not only to insects, but also to fungi, bacteria, nematodes, mites, and the like. The corn plants of the present invention are preferably resistant to an insect or insects selected from the group including, but not limited to, Ostrinia nubilalis, European corn borer; Sesemia nonegrioides, corn stem borer; Agrotis Ípsilon, black-cut worm; Helicoverpa zea, corn earworm, - Spodoptera frugiperda, autumn worm; Diatraea grandiosella, southwest corn borer Elasmopalpus lignosellus, minor corn stem borer; Diatraea saccharalis, sugarcane borer, Diabrotica virgif ra virgifra, western corn rootworm; Diabrotica longicornis barberi northern corn rootworm; Diabrotica undec impune tata howardi, southern corn rootworm; Melanotus spp. , wire worms; Cyclocephala borealis, northern masked bumblebee (white worm); Cyclocephala immaculata, southern masked bumblebee (white worm); Popillia japonica, Japanese beetle (worm and adult forms); Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, corn mincer; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinche; Melanoplus femurrubrum, red-footed grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, corn seed moth; Agromyza parvicornis, leaf mincer stained with corn; Anaphothrips obscurus, Thysanoptera _ of grass; Solenopsis miles ta, thief ant, and Tetranychus urticae, two-spotted spider mite. The sorghum plants of the present invention are preferably resistant to an insect or insects selected from the group including, but not limited to, Chilopartellus, sorghum borer; Spodoptera frugiperda, autumn worm; Helicoverpa zea, corn cob worm; Elasmopalpus lignosellus, minor corn stem borer; Fel tia subterranean, granulated cutworm; Phyllophaga crini ta, white worm; Eleodes, Conoderus, and Aeolus spp. , wire worms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, corn mincer; Rhopalosiphum maidis, corn leaf aphid; Sipha fiara, yellow aphid of sugarcane; Blissus leucopterus leucopterus, chinche; Contarinia sorghicola, sorghum gnat; Tetranychus cinnabarinus, carmine spider mite; and Tetranychus urticae, two-spotted spider mite. The wheat plants of the present invention are preferably resistant to an insect or insects selected from the group including, but not limited to, Pseudaletia unipunctata, worm worm; Spodoptera frugiperda, autumn worm; Elasmopalpus lignosellus, minor corn stem borer; Agrotis orthogonia, worm of pale western cut; Oulema melanopus, cereal leaf beetle; Hypera punctata, cloverleaf weevil; Diabrotica undec impune tata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, green bug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, red-footed grasshopper; Melanoplus differentialis differential grasshopper; Melanoplus sanguinipes, migratory skipjack; Mayetiola destroyer Hessian fly; If todiplosis mosellana, wheat midge; Meromyza americana, wheat stem moth; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thysanoptera; Cephus c inc tus, wheat stem sawfly; y Acería tulipae, wheat curly mite. The rice plants of the present invention are preferably resistant to an insect or insects selected from the group including, but not limited to, Diatraea saccharalis, sugar cane borer; Spodoptera frugiperda, autumn worm; Helicoverpa zea, corn cob worm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Si tophilus oryzae, rice weevil; Nephotettix nigropictus, rice grasshopper; Blissus leucopterus leucopterus, chinche; and Acrosternum hilare, green wood bug. The barley plants of the present invention are preferably resistant to an insect or insects selected from the group including, but not limited to, Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, green bug; Blissus leucopterus leucopterus, chinche; Acrosternum hilare, green wood bug; Euschistus servos, wooden cat bed bug; Hylemya platura, corn seed moth; Mayetiola destroyer Hessian fly; Thysanoptera, Thysanoptera; and Petrobia latens, brown-white mite. In one embodiment, the present invention relates to a transgenic monocotyledonous (monocotyledonous) plant, comprising recombinant DNA comprising a coding sequence that encodes peroxidase. The monocotyledons are plants whose embryo has a cotyledon. The monocotyledons are one of the two great classes of angiosperms (the dicotyledons being the other great class). Preferred families within the monocot class include: grasses (grass family; preferred members of grasses include forage grasses) (for example Festuca (cañuelas)), Hordeum (barley), Oats (oats), Zea mays (corn), Tri ticum, (wheat), Sécale (rye), Sorgum vulgare (sorghum), and Oryza sativa (rice)); lilaceae (family of lilacs, preferably Allium (onion) and Asparagus); and Dioschoceaceas (family of the yam), all of which are encompassed by the present invention. The present invention also includes, but is not limited to, the monocotyledonous species, for example, the Zea mays preferred lines include Funk 5N984, Funk 5N986, Funk 2717, Funk 211D, Funk 2N217A, B73, A632, CM105, B37, B84. , B14, Mol7, A188, CG00526, CG00615 and CG00714. The genetic properties designed in the seeds and transgenic plants mentioned above, are transmitted through sexual reproduction or vegetative growth, and therefore, can be maintained and propagated in descending plants. In general, this maintenance and propagation makes use of known agricultural methods developed to suit specific purposes, such as tillage, sowing, or harvesting. You can also apply specialized processes, such as hydroponics or greenhouse technologies. Since the growing crop is vulnerable to attack and damage caused by insects or infections, as well as competition with grass plants, measures are taken to control weeds, plant diseases, insects, nematodes, and other adverse conditions for improve performance. These include mechanical measures, such as tillage of the land or removal of infected plants and herbs, as well as the application of agrochemicals, such as herbicides, fungicides, gametocides, nematicides, growth regulators, ripening agents, and insecticides. . The use of the suitable genetic properties of the transgenic plants and seeds according to the invention, can be further made in the reproduction of plants which has as its objective the development of plants with better properties, such as tolerance to pests, to herbicides, or to the tension, better nutritional value, higher performance, or better structure that causes less loss of accommodation or collapse. The different reproduction steps are characterized by a well defined human intervention, such as selecting the lines to be crossed, directing the pollination of the parental lines, or selecting appropriate descendant plants. Depending on the desired properties, different reproduction measures are taken. Relevant techniques are well known in the art, and include, but are not limited to, hybridization, inbred reproduction, backcrossed reproduction, multiple line reproduction, variety blending, interspecific hybridization, aneuploid techniques, and the like. Hybridization techniques also include the sterilization of plants to produce male or female sterile plants by mechanical, chemical, or biochemical elements. The cross-pollination of a sterile male plant with pollen from a different line ensures that the genome of the male sterile but fertile female plant uniformly obtains the properties of both parental lines. Accordingly, seeds and transgenic plants according to the invention can be used for breeding of improved plant lines which, for example, increase the effectiveness of conventional methods, such as treatment with herbicide or pesticide, or allow to eliminate these methods due to to its modified genetic properties. Alternatively, new crops with better stress tolerance can be obtained which, due to their optimized genetic "equipment", produce a harvested product of better quality than the products that were not able to tolerate comparable adverse development conditions. In the production of seeds, the quality and uniformity of germination of the seeds are essential characteristics of the product, while the quality and uniformity of germination of the seeds harvested and sold by the farmer are not important. Since it is difficult to keep a crop free of other crop and herbal seeds, to control seed diseases, and to produce seeds with good germination, seed producers who are experienced in the cultivation, conditioning, and marketing of pure seeds, have developed very extensive and well-defined seed production practices. Therefore, it is common practice for the farmer to buy certified seeds that meet specific quality standards, instead of using seeds harvested from his own crop. The propagation material to be used as seeds is customarily treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematocides, molluscicides, or mixtures thereof. The customary protective coatings include compounds such as captan, carboxy, thiram (TMTD®), metalaxyl (Apron®), and pirimiphos-methyl (Actellic®). If desired, these compounds are formulated together with additional vehicles, surfactants, or auxiliary application promoters employed by the custom in the art of the formulation to provide protection against damage caused by bacterial, fungal, or animal pests. The protective coatings can be applied by impregnating the propagation material with a liquid formulation, or by coating it with a combined wet or dry formulation. Other methods of application are also possible, such as treatment directed towards shoots or fruit. It is a further aspect of the present invention to provide new agricultural methods, such as the methods exemplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seeds in accordance with the present invention.

In another embodiment, the present invention relates to a cell, tissue, organ, seed, or part of a transgenic plant, obtained from the transgenic plant.

Also included within the invention are the transgenic descendants of the plant, as well as cells, tissues, organs, seeds, and parts of transgenic plants obtained from the offspring. As described herein, the present invention relates to a fertile transgenic monocot plant transformed with a peroxidase coding sequence. Preferably, the peroxidase coding sequence confers on the monocot plant a phenotypic trait that is not found in a parent plant that lacks the peroxidase coding sequence or its overexpression. The phenotypic traits that can occur include insect resistance and the best chance of standing. More preferably, the peroxidase coding sequence in the transgenic plant is sexually transmitted. In a preferred embodiment, the peroxidase coding sequence is sexually transmitted through a complete normal sexual cycle from the RO plant to the Rl generation. In a further preferred manner, the peroxidase coding sequence is expressed in such a manner as to increase the level of peroxidase in cells, tissues, seeds, or plants, above the level of the cells, tissues, seeds, or plants of a monocot. that only differs in that the peroxidase coding sequence is absent. In a preferred embodiment, the peroxidase coding sequence is an anionic, cationic, or neutral peroxidase coding sequence. In another preferred embodiment, the peroxidase is a guaiacol peroxidase, NADH peroxidase, cytochrome-C peroxidase, catalase, glutathione peroxidase, L-ascorbate peroxidase, manganese peroxidase, hydrogen peroxide that generates peroxidase, and / or peroxidase lignin former. There are a variety of peroxidase coding sequences available in the art, and are available for use in the present invention. For example, peroxidases have been cloned from tobacco (Lagrimini, M., et al., Proc. Nati, Acad. Sci. USA 84: 7542-7546 (1987)), potato (Roberts et al., Plant Molecular Biology 11: 5-26 (1988)), radish (Fujiyama et al., European Journal of Biochemistry, 173 681-687 (1988), Fujiyama et al., Gene 89: 163-169 (1990), and Welinder, KG, European Journal of Biochemistry. 96: 483-502 (1979)), tomato (Roberts, E. and Kolattukudy, PE, Molecular Genes and Genetics, 217, 223-232 (1989)), peanut (Buffard et al., Proc. Nati, Acad. Sci. USA 87: 8874-8878 (1990)), cucumber (Morgens et al. Plant Molecular Biology 14: 115-125 (1990)), Arabidopsis (Intapruk et al., Gene 98: 231-241 (1991)), wheat (Hertig et al., Plant Molecular Biology 16.-171-174 (1991); and Rebmann et al., Plant Molecular Biology 16": 329-331 (1991)), barley (Rasmussen et al., Plant Molecular Biology 16: 311- 321 (1991); and Theilade, B. and Rasmussen, SK, Gene 118: 261-266 (1992)), rice (Reimman et al., Plant Physiology 100: 1611-1612 (1992)), corn (Thesis of Hwang, Ph.D., Ohio State University), and turnip (Mazza and Welinder, European Journal of Biochemistry 108: 481-489 (1980)). The peroxidase coding sequences used in the present invention should not be limited to the known peroxidase coding sequences. Novel peroxidase coding sequences can be isolated for use in the invention by identity or similarity with known sequences. The anionic peroxidase of tobacco shows identity or similarity with the amino acid sequences of cationic horseradish peroxidase and cationic turnip peroxidase. The overall identity or similarity between tobacco and radish is 52 percent; for tobacco and turnip, peroxidases have an identity or similarity of 46 percent. In addition, there are regions in the peroxidase coding sequence, in which the identity or similarity approaches 100 percent. Four of these conserved regions correspond to the critical domains for peroxidase activity in general. Accordingly, DNA sequences can be used from the conserved regions to generally clone the peroxidase coding sequences from any plant species, using methods well known in the art (see, for example, Current Protocols in Molecular Biology, eds: Ausubel et al., John Wiley &Sons, Inc. New York, NY (Spring 1996)). In the same way, novel peroxidase coding sequences can be isolated using antibodies made against a peroxidase enzyme to isolate other peroxidase enzymes. The homology between the different isozymes peroxidase with antibodies made for the anionic peroxidase of tobacco has been demonstrated. By Immunobloting analysis, these antibodies cross-reacted very strongly with the radish and turnip isozymes, and also cross-reacted with most of the other isozymes of tobacco. See Lagrimini, M., et al., Proc. Nati, Acad. Sci. USA 84: 7542-7546 (1987). The novel peroxidase enzymes can be sequenced using methods well known in the art, and their corresponding coding sequences can be isolated using methods well known in the art (for example, see Sambrook et al., Molecular Cloning-A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York, USA (1988)). The transgenic expression in plants of peroxidase coding sequences derived from different sources of the host plant (for example, from bacterial sources), may require the modification of these coding sequences to achieve and optimize their expression in the host plant. . In some cases, modification to the coding sequences and the adjacent sequence will not be required. It is sufficient to isolate a fragment containing the coding sequence of interest, and insert it downstream of a plant promoter. For example, see Gaffney et al., Science 261: 754-756 (1993). Preferably, very little adjacent microbial sequence must be left attached upstream of the ATG and downstream of the stop codon. The peroxidase coding sequence can be optimized for higher expression in the host monocot. For example, since the use of preferred codons and the codon frequency in the host plant may differ from the use and frequency of the peroxidase coding sequence of interest, the comparison of codon usage and frequency within a coding sequence cloned with the use and frequency in coding sequences of plants (and in particular coding sequences of the target plant) makes it possible to identify the codons within the coding sequence that can be changed in a preferable manner. Preferred codons can be determined from the higher frequency codons in the proteins expressed in greater amount in the plant, or from the more preferred codons in the plant. See, for example, Adang et al., European Patent Number EP-A-359, 472, Fischhoff et al., European Patent Number EP-A-385, 962, - Cornelissen et al., International Publication Number WO 91/16432.; Koziel et al., International Publication Number WO 93/07278; Perlak et al., Proc. Nati Acad. Sci. USA 88: 3324-3328 (1991); and Murray et al., Nucleic Acids Research 17: 477-498 (1989). In this way, the nucleotide sequences can be optimized for expression in the specific plant of interest. It is recognized that all or any part of the coding sequence can be optimized or synthesized. That is, synthetic or partially optimized sequences can also be used. Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Instead, the ribosomes bind to the 5 'end of the message, and look for the first available ATG in which to start the translation. In plants, there is a preference for certain nucleotides adjacent to the ATG, and therefore, the expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation primer in the ATG. Clontech (catalog 1993/1994, page 210) has suggested a sequence as a consensus translation primer for the expression of the uidA gene of E. coli in plants. Further, Joshi, Nucí. Acid Res. 15: 6643-6653 (1987) has compared many sequences of plants adjacent to the ATG, and also suggests a consensus sequence. In situations where difficulties are encountered in the expression of the microbial coding sequences in plants, the inclusion of one of these sequences in the starting ATG is preferred. Coding sequences cloned from sources other than plants may also contain motifs that can be recognized in plants as the 5 'or 3' splice sites, thereby generating truncated or suppressed messages. These sites can be removed using techniques known in the art (see, for example, Current Protocols in Molecular Biology, eds: Ausubel et al., John Wiley and Sons, Inc., New York, NY (Spring 1996)). A recombinant DNA comprising a coding sequence encoding a peroxidase can be used to produce transgenic plant tissues. A plant preferably is transformed with at least one recombinant DNA, which may further comprise a transcription initiation region and a promoter, both of which are operably linked to the peroxidase coding sequence. The transcription initiation regions may be native or foreign to the host. Strange means that the transcription initiation region is not found in the wild-type host where the transcription initiation region is introduced. The termination region can be obtained from: (1) the same gene from which the transcription initiation region was obtained, (2) the peroxidase gene used, or (3) derived from another source. The peroxidase coding sequence is preferably operably fused with a promoter that can be expressed in plants; Preferred promoters include constitutive, inducible, temporarily regulated, developmentally regulated, chemically regulated, tissue-preferred, and / or tissue-specific promoters. In a preferred embodiment, the peroxidase coding sequence is operably linked to its naturally occurring promoter, and / or to the sequence of the polyadenylation signal. Preferred constitutive promoters include the CaMV 35S and 19S promoters (Fraley et al., U.S. Patent No. 5,352,605). An additionally preferred promoter is derived from any of the different actin genes, which are known to be expressed in most cell types. The expression cassettes of the promoter described by McElroy et al., Mol. Gen. Genet. 231: 150-160 (1991) can be easily modified for the expression of the peroxidase coding sequence, and are particularly suitable for use in monocotyledonous hosts. Yet another preferred constitutive promoter is derived from ubiquitin, which is another genetic product that is known to accumulate in many cell types. The ubiquitin promoter has been cloned from several species for use in transgenic plants (eg, sunflower - Binet et al., Plant Science 79: 87-94 (1991), Christensen corn and coworkers, Plant Molec. 632 (1989)). The corn ubiquitin promoter has been developed in transgenic monocotyledonous systems, and its sequence and the vectors constructed for the transformation of monocotyledons are disclosed in Christiansen et al., European Patent Number EP-A-342, 926. The ubiquitin promoter is suitable for the expression of the coding sequence for peroxidase in transgenic plants, especially monocotyledons. The tissue-specific or tissue-preferential promoters useful for the expression of the peroxidase coding sequence in plants, particularly maize, are those that direct expression in the root, in the marrow, in the leaf, or in the pollen. These promoters are disclosed in Koziel et al., International Publication Number WO 93/07278. Chemically inducible promoters useful for directing the expression of the peroxidase coding sequence in plants are also preferred (see Alexander et al., International Publication Number WO 95/19443).

In addition to the promoters, a variety of transcription terminators are also available for use in the construction of chimeric genes using a peroxidase coding sequence. Transcription terminators are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Suitable transcription terminators, and those known to work in plants, include the CaMV 35S terminator, the tml terminator, the rbcS E9 pea terminator, and others known in the art. There are convenient termination regions available from the Ti plasmid of A. tumefaciens, such as the opalpine synthase and nopaline synthase termination regions. See also Rosenberg et al., Gene 56: 125 (1987); Guerineau and collaborators Mol. Gen Genet , 262: 141-144 (1991); Proudfoot, Cell, 64: 611-614 (1991); Sanfacon and collaborators Genes Dev., : 141-149; Mogen et al., Plant Cell, 2: 1261-1212 (1990); Munroe et al., Gene, 91: 151-158 (1990); Bailas et al. Nucleic Acids Res. 17: 7891-7903 (1989); Joshi et al. Nucleic Acid Res. , 15: 9621-9639 (1987)). It has also been found that numerous sequences improve gene expression from within the transcription unit, and these sequences can be used in conjunction with the peroxidase coding sequence to increase expression in transgenic plants. It has been shown that different sequences of introns improve expression, particularly in monocotyledonous cells. For example, it has been found that the introns of the Adhl corn gene significantly increase the expression of the wild-type gene under its known promoter when introduced into corn cells (Callis et al., Genes Develop 2: 1183-1200 ( 1987)). Intron sequences have been routinely incorporated into plant transformation vectors, usually within the non-translated leader. The construct may also include a regulator, such as a nuclear localization signal (Kalderon et al., Cell 39: 499-509 (1984); and Lassner et al., Plant Molecular Biology 17: 229-234 (1991)), a sequence translational consensus plant (Joshi, CP, Nucleic Acids Research 15: 6643-6653 (1987)), an intron (Luehrsen and Walbot, Mol. Gen. Genet 225: 81-93 (1991)), and the like, operatively linked with the appropriate nucleotide sequence. Preferably, the 5F leader sequence is included in the construction of the expression cassette. These leader sequences can act to improve translation. Translational leaders are known in the art, and include: leaders of picornavi-rus, for example the leader EMCV (5F nonencoding region of encephalomyocarditis) (Elroy-Stein, O., Fuerst, TR, and Moss, Proc. Nati. Acad. Sci. USA 86: 6126-6130 (1989)); Potivirus leaders, for example the TEV leader (Tobacco Engraving Virus) (Allison and collaborators, MDMV leader (Corn Dwarf Mosaic Virus), Virology 154: 9-20 (1986)), and human immunoglobulin heavy chain binding (BiP) protein (Macejak, DG and Samow, P., Nature 353: 90-94 (1991), untranslated leader from the protein mRNA of alfalfa mosaic virus coating (AMV RNA 4) (Jobling, S.A., and Gebrke, L., Nature, 325: 622-625 (1987)); tobacco mosaic virus leader (TMV) (Gallie, D.R. et al., Molecular-Biology of RNA, pages 237-256- (1989)); and leader of corn chlorotic spotted virus (MCMV) (Lom-mel, S.A. et al., Virology 51: 382-385 (1991)). See also, Della-Cioppa et al., Plant Physiology 84: 965-968 (1987). In the preparation of the recombinant DNA, different DNA fragments can be manipulated, to provide the DNA sequences in the proper orientation, and as appropriate, in the appropriate reading frame. Towards this end, adapters or linkers can be used to join DNA fragments, or other manipulations can be used to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, preference is given to in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like, where insertions, deletions, or substitutions are involved, for example, transitions and transitions. There are numerous transformation vectors available for the transformation of plants, and the coding sequences for peroxidase can be used in conjunction with any of these vectors. The selection of a vector to be used will depend on the preferred transformation technique and the target species for the transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. The selection markers routinely used in the transformation include the nptll gene that confers resistance to kanamycin and related antibiotics (Messing & amp;; Vierra, Gene 19.-259-268 (1982); Bevan et al, Nature 304: 184-181 (1983)), the jbar gene that confers resistance to the herbicide phosphinothricin (White et al., Nucí Acids, Res., 18: 1062 (1990), Spencer and collaborators Theor. Appl. Genet 75: 625-631 (1990)), the hph gene that confers resistance to the antibiotic hygromycin (Blochinger &Diggelmann, Mol Cell. Biol. 4: 2929-2931), and the dhfr gene, which confers resistance to methotrexate ( Bourouis et al., EMBO J. 2: 1099-1104 (1983)). There are many vectors available for transformation using Agrobacterium tumefaciens. These normally carry at least one T-DNA limit sequence, and include vectors such as pBIN19 (Bevan, Nucí Acids, Res. 12 (22): 8711-8721). (1984) ) . In a preferred embodiment, the peroxidase coding sequence can be inserted into any of the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium These vector cassettes for Agrobacterium-mediated transformation can be constructed in the following manner. PTJS75kan was created by digestion with NarI from pTJS75 (Schmidhauser &Helinski, J "Bacteriol., 164: 446-455 (1985)), allowing separation of the tetracycline resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al. Plant Molecular Biology 14: 266-276 (1990). Xhol linkers were ligated to the EcoRV fragment of pCIB7 containing the boundaries of left and right T-DNA, a chimeric us / nptll gene selectable in plants, and the pUC polylinker (Rothstein et al. Gene 53: 153-161 (1987)), and the fragment digested with XhoI was cloned into pTJS75kan digested with SalI to create pCIB200 (see also Patent European Number EP-A-332, 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, Kpnl, BglII, Xbal, and Salí pCIB2001 is a derivative of pCIB200 that was created by inserting additional restriction sites into the polylinker. The unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, Kpnl, BglII, Xbal, Salí, MIuI, BcII, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites, also has kanamycin selection in plants and bacteria, left and right T-DNA boundaries for Agrobacterium-mediated transformation, the function of trfA derived from RK2 for mobilization between E coli and other hosts, and the functions of OriT and OriV also from RK2. The polylinker pCIB2001 is suitable for the cloning of expression cassettes in plants that contain their own regulatory signals. An additional vector useful for Agrobacterium-mediated transformation is the binary vector pCIBlO which contains a gene coding for kanamycin resistance for plant selection, the right and left T-DNA border sequences, and incorporates sequences from the plasmid of a wide range of hosts pRK252, which allows it to replicate in both E. Coli and Agrobacterium. Its construction is described by Rothstein et al., Gene 53: 153-161 (1987). Different pCIBlO derivatives have been constructed that incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. Gene 25: 179-188 (1983). These derivatives make it possible to select cells from transgenic plants on hygromycin alone (pCIB743), or on hygromycin and kanamycin (pCIB715, pCIB717). One of these vectors useful for direct gene transfer techniques in combination with the selection by the herbicide Basta (or phosphinotricin) is pCIB3064. This vector is based on the plasmid pCIB246, which comprises the 35S promoter of CaMV in fusion operative with the GUS gene of E. coli and the transcription terminator 35S of CaMV, and is described in Koziel et al., International Publication Number WO 93/07278. The gene that provides resistance to phosphinothricin is the gene from Streptomyces hygroscopicus (Thompson et al., EMBO J. 6 ': 2519-2523. (1987)). This vector is suitable for the cloning of expression cassettes in plants that contain their own regulatory signals. An additional transformation vector is pSOG35, which uses the dihydrofolate reductase (DHFR) of the E. coli gene as a selectable marker, which confers resistance to methotrexate. Polymerase chain reaction was used to amplify the 35S promoter (approximately 800 base pairs), intron 6 from the Adhl maize gene (approximately 550 base pairs, see Dennis et al.

Nucleic Acid Res. 12: 3983-4000 (1984)), and 18 base pairs of the GUS untranslated leader sequence (see Jefferson et al., Proc. Nat. Acad. Sci. USA 83: 8441- 8451 (1986)).

A 250-base pair fragment of the gene encoding the type II dihydrofolate reductase from E. coli was also amplified by polymerase chain reaction, and these two fragments obtained from the polymerase chain reaction were assembled with a SacI fragment. -PstI from pBI221 (Clontech), which comprised the base structure of the pUC19 vector, and the nopaline synthase terminator. The assembly of these fragments generated pS0G19, which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the gene DHFR, and the nopaline synthase terminator. The replacement of the GUS leader in pS0G19 with the leader sequence of the Speckled Virus Chlorotic Corn (MCMV), generated the vector pSOG35. pS0G19 and pSOG35 carry the pUC gene for ampicillin resistance, and have the HindIII, Sphl, PstI, and EcoRI sites available for the cloning of foreign sequences. The recombinant DNA described above can be introduced into the plant cell in a number of ways recognized in the art. Those skilled in the art will appreciate that the choice of method could depend on the type of plant to be used for the transformation. Suitable methods for transforming plant cells include microinjection (Crossway et al., Bio Techniques 4: 320-334 (1986)), electroporation (Riggs et al., Proc. Nati.

Acad. Sci. USA 83: 5602 -5606 (1986)), Transformation mediated by Agrobacterium (Hinchee et al., Biotechnology 6: 915-921 (1988); See also Ishida et al, Nature Biotechnology 14: 145-150 (June 1996) for the transformation of corn), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984); Hayashimoto et al., Plant Physiol. 93 - 851- 863 (1990) (rice)), and acceleration of ballistic particles using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent No. 4,945,050, and McCabe et al., Biotechnology 6: 923 -926 (1988)). See also, Weissinger et al., Annual Rev. Genet 22: 421-477 (1988); Sanford et al., Particulate Science and Technology : 21-31 91987) (onion); Svab et al., Proc. Nati Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al., Plant Physiol. 87: 611-614 (1988) (soy bean); McCabe et al., Bio / Technology 6: 923-926 (1988) (soybean); Klein et al., Proc. Nati Acad. Sci. USA 85: 4305-4309 (1988) (corn); Klein and collaborators, Bio / Technology 6 \ -559-563 (1988) (corn); Klein and collaborators, Plant Physiol. 51: 440-444 (1988) (corn); Fromm and collaborators, Bio / Technology 8: 833-839 (1990); and Gordon-Kamm and collaborators, Plant Cell 2: 603-618 (1990) (corn); Koziel et al Biotechnology 21: 194-200 (1993) (corn); Shimamoto et al.

Nature 338: 274-277 (1989) (rice); Christou and collaborators, Biotechnology 9: 951-962 (1991) (rice); Datta and collaborators, Bio / Technology 8: 136-140 (1990) (rice); Patent request European Number EP-A-332.581 (orchard grass and other Pooideae); Vasil et al. Biotechnology 11: 1553-1558 (1993) (wheat); Weeks and collaborators, Plant Physiol. 102: 1011-1084 (1993) (wheat); Wan and collaborators, Plant Physiol. 104: 31-48 (1994) (barley); Jahne and collaborators Theor. Appl.

Genet 85: 525-533 (1994) (barley); Umbeck and collaborators, Bio / Technology 5: 263-266 (1987) (cotton); Casas y colaboradores, Proc. Nati Acad. Sci. USA 90: 11212-11216 (December 1993) (sorghum); Somers and collaborators, Bio / Technology 10: 1589-1594 (December 1992) (oats); Torbert et al., Plant Cell Reports 14: 635-640 (1995) (oats); Weeks and collaborators, Plant Physiol. 102: 1011-1084 (1993) (wheat); Chang and collaborators, International Publication Number WO 94/13822 (wheat) and Nehra et al., The Plant Journal 5: 285-291 (1994) (wheat). A particularly preferred set of embodiments for the introduction of recombinant DNA molecules into corn by bombardment of microprojectiles can be found in Koziel et al., Biotechnology 12: 194-200 (1993), Hill et al., Euphytica 85: 119-123 ( 1995) and Koziel et al., Annals of the New York Academy of Sciences 752: 164-171 (1996). A further preferred embodiment is the protoplast transformation method for corn, as disclosed in Shillito et al., European Patent Number EP-A-292,435. The transformation of the plants can be undertaken with a single DNA species, or with multiple DNA species (i.e., co-transformation), and both techniques are suitable for use with the peroxidase coding sequence.

Methods that use either a form of direct gene transfer, particle gun technology, or transfer measured by Agrobacterium, normally, but not necessarily, are undertaken with a selectable or classifiable marker that provides resistance to an antibiotic (eg, kanamycin, hygromycin, or methotrexate) or a herbicide (eg, phosphinothricin). Examples of these labels are neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2,2-dichloropropionic acid dehalogenase, acetohydroxy acid synthase, 5-enolpyruvyl-shikimate phosphate synthase, haloarylnitrilase, carboxylase acetyl-coenzyme A, dihydropteroate synthase, chloramphenicol acetyltransferase and β-glucuronidase. However, the choice of selectable or classifiable marker for transformation into plants is not critical to the invention. The peroxidase coding sequence is preferably used alone or in combination. That is, one or more peroxidase coding sequences can be inserted into a plant to control different insect pests. This can be done by: (1) transforming a host plant with a DNA sequence comprising more than one peroxidase coding sequence, (2) transforming a host plant with a DNA sequence comprising a single coding sequence of peroxidase, and the identification of multiple insertions of the DNA sequence in the host genome or (3) the repeated transformation of a host plant with a peroxidase coding sequence, until the host plant comprises the desired number of peroxidase coding sequences . The level of insect protection of a plant against a given insect and / or its spectrum of insecticidal activities can also be increased by combining a peroxidase-coding sequence with other coding sequences that encode proteins capable of controlling insects. Bacillus thuringiensis (Bt) is a gram-positive spore-forming bacterium that produces a parasporal crystal during sporulation (for a review see Koziel et al, Biotech and Gen. Reviews 22: 171-228 (1993)). These crystals are predominantly comprised of one or more proteins, termed d-endotoxins or insecticidal crystal proteins, which are known to possess insecticidal activity when ingested by certain insects. Numerous strains of Bt are currently known. Each strain produces different numbers of d-endotoxins with different insecticidal activities. Examples of Bt endotoxins that can be used in combination with peroxidases include, but are not restricted to, Cry? A (b) (Koziel et al., Bio / Technology 22: 194-200 (1993)), Cry? A (c) (U.S. Patent No. 5,530,197), CrylH (also referred to as Cry9C) (Lambert et al., Environ Microbiol., 62: 80-86 (1996)), and CrylIA (Adang et al., Plant Mol. Biol. 21: 1131-1145 (1993)). Pesticide proteins produced during the vegetative growth of the Bacillus strains (vegetative insecticidal proteins, VIPs) in combination with peroxidases can also be used. For examples of VIPs see Warren et al., International Publication Number WO 94/21795; Warren et al., International Publication Number WO 96/10083; and Estruch et al., Proc. Nati Acad. Sci. USA 93: 5389-5394 (1996). Examples of other proteins with insecticidal compounds that can be used in combination with peroxidases include, but are not restricted to, cholesterol oxidases (U.S. Pat. ,518,908), protease inhibitors, lectins, and α-amylases. Monocotyledons expressing more than one insect resistance coding sequence can be made by any method known in the art. For example, the peroxidase coding sequence can be used to transform a monocot at the same time as another insect principle gene (cotransformation); the second insect principle gene can be introduced into a plant that has already been transformed with a peroxidase coding sequence, or vice versa, or alternatively, transgenic plants can be crossed, one expressing a peroxidase coding sequence and one that expresses a second insect principle, to gather the coding sequences in the same plant. The present invention is described in a further detail in the following non-limiting examples. In the examples, the procedures for manufacturing, manipulating, and analyzing nucleic acids are carried out by conventional methods, as described in Sambrook et al.

Molecular Cloning-A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA (1988).

EXAMPLES Example 1: Transgenic Corn Plants Comprising a Peroxidase Coding Sequence Construction of Vector pP0D3.s (Lagrimini et al., Proc.Nat.Acid.Sci 84: 438-442 (1987)) contains the anionic peroxidase cDNA of tobacco of 1256 base pairs, which comprises the entire peroxidase coding sequence, which includes a 22 amino acid signal peptide that facilitates secretion into the cell wall space. pP0D3.s was digested with BamHI, and cloned into the BamHI sites of pCIB710 (Rothstein et al., Gene 53: 153-161 (1987)). This new construct was digested with EcoRI, and subcloned into Bluescript SK + (Stratagene Catalog, 1994) - the resulting construction designated as pJS20293 (Figure 1) was put on deposit in the Agricultural Research Culture Collection (NRRL), International Depositary Authority, 1815 N. University Street, Peoria IL 61604 E.U.A., September 27, 1996, as NRRL B-21626. pJS20293 contains the 1200 base pair peroxidase cDNA clone behind the CaMV 35S promoter, and the shrunk intron (Werr et al., EMBO J. 4: 1373-1380 (1985)), followed by the 35S CaMV terminator in a BlueScript plasmid (Stratagene) (Figure 1) . pJS20293 was cotransformed with pUBI / Ac (Figure 2), a plasmid containing a chimeric bar gene that codes for phosphinothricin resistance.

Transformation Using Immature Cigotic Embryos In two separate experiments, 600 immature embryos of CG00526, a Lancaster-type inbred, 12 to 13 days after pollination, were cut aseptically from cobs grown in a surface-sterilized greenhouse. The embryos of a size of 1.5 to 20 millimeters were covered with the scutellum on a medium of initiation of callus, 2DG4 + 5 milligrams / liter of chloramben. The 2DG4 medium is Duncan's medium "D" (Duncan et al., Plant 165: 322-332 (1985)) modified to contain 20 milligrams / liter of glucose. The DNA of pJS20293 was precipitated on a 1-millimeter gold microcarrier, as described by the Dupont Biolistic manual. The DNA / gold mixture was prepared to apply about 1 milligram of pJS20293 DNA per bombardment. For the transformation of the immature embryo, 6.34 milligrams of pJS20293 + 7.21 milligrams of pUBl / Ac were used per 50 milliliters of microcarrier. Both preparations were taken up to 85 milliliters with ethanol, and 10 milliliters of each were dried on macrocarriers. Four hours before the bombardment, the embryos were transferred to 12DG4 + 5 milligrams / liter of chloramben for osmotic treatment. 36 embryos were bombarded on a plate using the PDS-100OO Biolistics device according to the manufacturer's instructions (Dupont). The embryos were accommodated on the target plate around a circle 2 centimeters in diameter in the center of the plate, with the coleorrhiza ends of the escutelo all oriented in the same direction. Then the objective plates were angled upwards at an angle of 30 ° C, such that the coleorrhizal ends were first impacted by the particle spray. A standard 24 x 24 millimeter mesh supplied by the manufacturer Biolistic was used, with rupture discs with a value of 108.5 kg / cm2 for the bombings. Three hours after the bombardment, the embryos were returned to the medium of 2DG4 + 5 milligrams / liter of chloramben, and then they were grown in the dark at 25 ° C. Fourteen days after the bombardment, the embryogenic responses were transferred to a calliper maintenance medium of 2DM4 + 0.5 milligrams / liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The M4 medium is the same as G4 minus the casamino acids. This medium contained 5 milligrams / liter of Basta®, and gradually increased until milligrams / liter. Regeneration was started after 12 weeks on Basta® selection. Type I callus was subcultured on a modified Murashige and Skoog (MS) medium (Murashige and Skoog, Physiologia Plantarum 15: 413-491 (1962)) containing sucrose 3 percent, 0.25 milligrams / liter of 2,4-D, 5 milligrams / liter of benzylic aminopurine, and 5 milligrams / liter of Basta®, and cultured under 16 hours of light (50 mE / m-2 / sl) , 8 hours of darkness, at 25 ° C. Two weeks later, the tissue was transferred to an MS medium containing 3 percent sucrose and 5 milligrams / liter of Basta®. The regenerated plants were grown on a modified MS medium to contain half the salt concentration and 3 percent sucrose in GA7 containers.

Transformation Using Type I Embryogenic Callus For transformation of cusing type I embryogenic callus, callus was obtained from immature zygotic embryos using conventional culture techniques. For the delivery of the gene, approximately 300 milligrams of type I callus were prepared either by pricking with a scalpel blade, or subculturing 3 to 5 days prior to gene delivery. Before delivery of the gene, the prepared callus was placed on a semi-solid culture medium again containing 12 percent sucrose. After approximately 4 hours, the tissue was bombarded with pJS20293, using the PDS-1000 / He Biolistic device from BioRad. Two milligrams of pJS20293 were precipitated on 1-millimeter gold particles, essentially using the conventional BioRad protocol. Approximately 16 hours after the gene was delivered, the callus was transferred to a conventional culture medium containing 2 percent sucrose and 1 milligram / liter of phosphinothricin. The callus was subcultured on selection for 8 weeks, after which the surviving and growing callus was transferred to a standard regeneration medium for plant production. The regenerated plants were tested to determine their resistance to European cborer. Resistant plants were obtained. The resulting transgenic plants were used in a conventional plant breeding scheme to produce more transgenic plants with similar insecticidal properties. The transgenic plants were also crossed with other varieties of the same plant. The transgenic plants also produced seeds that contained the gene for chimeric peroxidase stably inserted in its genome. The transgenic cplants containing the peroxidase coding sequence were identified by Southern blot analysis. When the genomic DNA of the transgenic plants was digested with the restriction enzyme EcoRI, a band of approximately 1.3 Kb could be detected using the gene for peroxidase as a specific probe that indicated the presence of an intact peroxidase gene. The transgenic cexpressing the coding sequence for peroxidase was also identified by Northern blot. A band of approximately 1.2 kb in size was observed in the RNA stain when it was hybridized with a peroxidase-specific probe.

Insect Bioassay A total of 46 transgenic maize plants were originally evaluated to determine their insecticidal activity against Ostrinia nubilalis (ECB). This first bioassay group was performed by applying 10 ECB larvae in the first moult to a leaf cut that had been placed in a Gelman Petri dish with a moistened filter pad to prevent drying of the leaf cut. The larvae were allowed to feed without altering for 2 days. Two plants of event 554 were positive in the preliminary bioassay. These two plant samples showed no signs of insect feeding, and the larvae were dead. The rest of the plants of that event and other events had insects feeding healthy. With this activity observed, more repetitions were made in the following ECB bioassay. Four repetitions were made with 5 larvae per repetition. A mortality percentage reading was taken after two days. Since the plants still seemed to contain insecticidal activity, it was decided to test them against other target insects. Transgenic maize plants expressing the peroxidase enzyme were tested for their insecticidal activity by means of insect bioassays. The procedure is similar for any maize plant transformed with any insecticidal gene, but is described herein using as an example a coding sequence for peroxidase. One to four sections of four centimeters are cut from an extended leaf of a transformed corn plant. Each piece of sheet is placed on a filter disc moistened in a Petri dish of 50 x 9 millimeters. Five neonatal larvae of the target insect (European corn borer, autumn worm, corn cob worm, bean warrior worm, and black cut worm) were placed on each piece of leaf. Since each plant is sampled multiple times, this makes a total of 5 to 20 larvae per plant. The Petri dishes were incubated at 30 ° C, and damage was assessed by feeding the leaf and mortality data at 24, 48, and 72 hours. The toxicity data are shown in Table I. Table I; Mortality values of different insects when exposed to corn leaves that express the enzyme peroxidase. 554-1 554-3 Ostrinia nubilalis (ECB) 100% 100% Spodoptera frugiperda (FAW) 15% 15% Spodop t was exi gua (BAW) 0% 5% Heliothis zea (CEW) 100% 100% Agrotis ípsilon (BCW) 0% 0% These leaves had a strong anti-feeding effect against the autumn worm.

Transgenic Descendants The transgenic maize plants of event numbers 554 and 755 (which have been shown to have insecticidal properties against the European corn borer under in vitro bioassays) were field tested. When the plants in the field reached approximately 40 centimeters of height of the extended leaf, the infestations with ECB larvae began. Approximately 300 neonatal larvae mixed with corn cob grits were deposited in the whorl of each plant. The infestations continued on a weekly basis for 4 weeks to simulate the first generation corn borer (ECB1). Starting two weeks after the initial infestation, each plant was evaluated weekly, and an average damage assessment of ECB1 was scored (see tables II and III). When the corn plants reached the anthesis, 300 neonatal larvae per plant were applied weekly for 4 weeks to simulate the infestation of the second generation (ECB2). Approximately 50 days after the initial ECB2 simulated infestation, the stems were divided, and tunnel damage was measured (see Tables II and III). The experimental conditions are further detailed by Koziel et al. Bio / Technology 12: 194-200 (1993).

The evaluations of leaf damage were determined as follows: 1. There is no visible lesion of the leaf 2. Evidence of fine "window" damage only on the unrolled leaf where the larvae plus the corn cob grits fell on the whorl . There is no penetration of holes in the blade. 3. Evidence of fine "window" damage on two uncoiled leaves, where the larvae plus the corn cob grit fell on the whorl. There is no penetration of holes in the blade. 4. Evidence of hole or damage by feeding of perforated orifice that penetrated the leaf, in two or more leaves that emerged from the whorl. (any lesion <0.635 centimeters in length). 5. Elongated lesions and / or feeding of the midrib evident in more than 3 leaves that emerged from the whorl. Injuries < 2.54 centimeters in length. 6. Several leaves with elongated lesions (from 1,905 centimeters to 3.81 centimeters in length), and / or no more than one leaf with a broken midrib. 7. Long lesions (> 2.54 centimeters) common in approximately half of the leaves, and / or two or three leaves with broken midribs. 8. Long lesions (> 2.54 centimeters) common in approximately 2/3 parts of the leaves and / or more than 3 leaves with broken midribs. 9. Most leaves with long lesions. Several leaves with half-broken ribs. Possibly atrophied plants due to the feeding of ECB. 2The extension of the internal tunneling damage by ECB in a section of 92 centimeters of the stem, 46 centimeters above and below the primary node of the ear, was measured in transgenic and control plants. The maximum damage that can be evaluated is 92 centimeters. The control plants were completely destroyed at the end of the experiment, and therefore, no measurement was possible.

The foliar damage evaluations were determined as described for Table II: 1. There is no visible lesion of the leaf 2. Evidence of fine "window" damage only on the unrolled leaf where the larvae plus the cob grit corn fell in the whorl. There is no penetration of holes in the blade. 3. Evidence of fine "window" damage on two uncoiled leaves, where the larvae plus the corn cob grit fell on the whorl. There is no penetration of holes in the blade. 4. Evidence of hole or damage by feeding of perforated orifice that penetrated the leaf, in two or more leaves that emerged from the whorl. (any lesion <0.635 centimeters in length). 5. Elongated lesions and / or feeding of the midrib evident in more than 3 leaves that emerged from the whorl. Injuries < 2.54 centimeters in length. 6. Several leaves with elongated lesions (from 1,905 centimeters to 3.81 centimeters in length), and / or no more than one leaf with a broken midrib. 7. Long lesions (> 2.54 centimeters) common in approximately half of the leaves, and / or two or three leaves with broken midribs. 8. Long lesions (> 2.54 centimeters) common in approximately 2/3 parts of the leaves and / or more than 3 leaves with broken midribs. 9. Most leaves with long lesions. Several leaves with half-broken ribs. Possibly atrophied plants due to the feeding of ECB. 2 The extension of internal tuning damage by ECB in a section of 92 centimeters of the stem, 46 centimeters above and below the primary node of the ear, was measured in transgenic and control plants. The maximum damage that can be evaluated is 92 centimeters. The control plants were completely destroyed at the end of the experiment, and therefore, no measurement was possible.

Example 2 Transgenic Wheat Plants Comprising a Peroxidase Coding Sequence pJS20293 (Figure 1) and pUBIAc (Figure 2) are used to transform wheat, using the methods of Chang et al., International Publication Number WO 94/13822, Weeks et al. , Plant Physiol 102: 1077-1084 (1993) or Nehra et al., The Plant Journal 5 (2). -285-291 (1994). The transformation of wheat using a method of Chang et al., International Publication Number WO 94/13822, is briefly stipulated as follows (other methods stipulated in Chang et al. May also be used): Preparation of Wheat Callus, Genotype UC703 Wheat plants of genotype UC703 are grown until flowering, and self-pollinated. The spikes containing embryos 1 to 2.5 millimeters long are removed from the plants, and sterilized with a 10 percent Clorox solution for 10 minutes. The embryos of the immature seeds are removed, and they are placed with the axis of the embryo down on the medium, on Murashige and Skoog containing 5 or 10 milligrams / liter of 2,4-D, maltose at 13.7 weight / volume, 100 milligrams / liter of proline, and 100 milligrams / liter of myoinositol solidified with phytagar at 0.7-0.8 volume / volume, or gelrite at 0.1-0.2 percent (initiation medium). After a three-week culture in the dark at 27 ° C, a callus is recognized as preferred by the presence of well-formed globular somatic embryos (callus type M) that develop on the scutellum of certain explants. These calluses are removed and placed either on an MS medium containing 1.0 to 5.0 milligrams / liter of 2,4-D and 2-3% sucrose, or on a medium containing a reduced level (5 percent) of maltose , before placing on the sucrose medium. Then the material is subcultured every week in fresh MS medium containing 3 percent sucrose.

Cellular Preparation For Bombardment The cells for bombardment are given a plasmolysis treatment before and after the bombardment. The packed cell volume is measured, and the cells are diluted in a 1 MS liquid medium with osmotic aggregate: 0.4 M sorbitol for the suspension cells, and 0.6 M sorbitol for the callus cells. The cells are diluted in such a way that the final packed cell volume per target is 1/30 milliliters for a fine suspension, and 1/10 milliliters for the callus. The diluted cells are placed in a 250 milliliter flask containing a stir bar, and stirred for a minimum of 30 minutes, for a few hours. To coat the cells, 2 milliliters are removed from the flask, and pipetted to the top of a flask in vacuo on which a Whatman 2.5 cm GFA filter is placed. The vacuum is applied until the cells are dried on the filter.

The filters are placed on Petri dishes of 60 x 15 millimeters containing 5 milliliters of solid plasmolysis medium after bombardment, which is 1 MS containing 0.2 M sorbitol for suspended cells, or 0.4 M sorbitol for callus cells. Two filters are coated on each disk.

Vectors Used for Bombardment The following vectors can be used in which an expression cassette containing peroxidase is inserted., for bombardment of particles (in addition to the cotransformation of pJS20293 (Figure 1) and pUBIAc (Figure 2), using equal amounts of DNA): pSOG30 is an expression vector of β-glucuronidase (Gus) derived from the plasmid pBI121, purchased from Clontech Laboratories, Palo Alto, California. The intron 6 of the maize Adh 1 gene is amplified by polymerase chain reaction from plasmid pB428, described in Bennetzen et al., Proc. Nati Acad. Sci. USA 81: 4125-4128 (1987), and ligated into the BamHI site of pBI121, which is between the 35S promoter of CaMV and the Gus gene. A leader of corn chlorotic mottled virus (MCMV) of 17 base pairs, described in Lommel et al., Virology 282: 382-385 (1991), is inserted in the 35S non-translated leader of the Gus gene. The final genetic fusion contains the structure: promoter 35S-intron 6 of Adhl-leader MCMV-Gus-Terminator Nos, all in the base structure of the pUC19 vector. pSOG35 is a dihydrofolate reductase (dhrf) expression vector. This construction is derived by fusion of the 35S promoter, the Adh 1 intron 6, and the MCMV leader described above, with the dhfr gene from the plasmid pHCO, described in Bourouis and Jarry, EMBO J. 2: 1099-1104 (1983). The final genetic fusion contains the structure: promoter 35S-intron 6 of Adh 1-leader MCMV-dhfr-terminator Nos, all in the base structure of the vector pUC19. pTG48 comprises the Gus gene under the control of the anther-specific promoter ant43D, and a dhfr gene in a base structure of pUC19. It is the result of the combination of 4 different fragments of DNA. Fragment 1 is obtained from pSOG35 after restriction cut with HindIII and EcoRI. The EcoRI end of the isolated fragment containing the dhfr gene is adapted to a SalI restriction end. Fragment 2 consisted of anther-specific promoter ant43D isolated from the plasmid pCIB3178 after restriction cleavage with HindIII and Xbal. Plasmid pCIB3178 is described in detail in European Patent Number EP-578,611, the relevant parts of which are incorporated herein by reference, and deposited under accession number NRRL B-18978.

The fragment 3 is obtained from the plasmid pSOG30 after the restriction cut with Xbal and EcoRI, and contained the gene Gus, and fragment 4 corresponded to the commercially available vector pUC19 cut with Sali and EcoRI.

Particle Preparation Gold particles (1.0 micron, from Bio-Rad) are washed by aliquoting in a microcentrifuge tube, adding approximately 1 milliliter of 100 percent ethanol, vortexing, centrifuging, removing the supernatant, and repeating twice. the sterile water. After the final wash, remove as much water as possible, and add a solution of polylysine (polylysine 0.02 percent plus 15 mM ammonium acetate) to completely submerge the particles. The particles are vortexed, centrifuged, and the supernatant is removed. The particles are allowed to dry overnight in a laminar flow hood, or for 30 minutes under a slight stream of nitrogen. For a preparation of "complete" particles, weigh 10 milligrams of particles and place them in a sterile microcentrifuge tube containing a stir bar. 100 milliliters (1 milligram / milliliter) of each DNA is added (alternatively, 50 milliliters (1 milligram / milliliter) of each DNA), followed by vortexing. Then 10 milliliters of 100 mM Na2HP04 are added, followed by vortexing. 10 milliliters of 100 nM CaCl2 are added, followed by a whirlpool.

Finally, 380 milliliters of 100% ethanol are added, followed by vortexing. While stirring the suspension vigorously, 3 milliliters are pipetted onto plastic ruffles (projectiles). The particles are allowed to dry on the flyers for at least 15 minutes before bombardment.

Bombardment of Cellular Cul tives The Petri box, which contains the cellular filters, is inverted on the platform above the stage, and centered on the particle flight aperture. The transparent cover is placed on top of the platform. A microprojectile is placed on the bolt of the stock, and the stock is closed. The "arm" button is pressed to fill the reservoir with the appropriate amount of helium gas (usually from 126 to 133 kg / cm2). The vacuum is pulled over the chamber to approximately 27 millimeters. After the vacuum is deactivated, the "arm" and "shoot" buttons are pressed. Then the "arm" button is pressed to the "off" position. Each filter is triggered normally twice.

Cul tive and Selection After Bombing After the bombing, the cells are kept in the dark at night. The next day, the filters of the plasmolysis medium are removed and placed on a 1 MS medium. The selection is applied 1 to 10 days after the bombardment for the cells in suspension, and after 14 days for the callus cells. The cells are scraped from the filters, and spread over the surface of the boxes containing 1 MS plus 2 milligrams / liter of methotrexate (or the appropriate selective agent). The boxes are incubated in the dark for several weeks. Resistant colonies that occur after a few weeks are transferred to 1 MS plus 4 milligrams / liter of methotrexate (or the appropriate selective agent). Colonies that continue to proliferate for approximately 3 to 4 weeks are then transferred to a maintenance medium "0.5 MS", which is an aqueous solution of MS salts, vitamins, iron, 3 percent sucrose, 0.7 percent agar , 0.5 milligrams / liter of 2,4-D. The tissue is subcultured on this medium fortnightly, until the embryogenic structures appear, or the tissue seems suitable for regeneration.

Regeneration The tissue is transferred to an MS medium containing either 3 milligrams / liter of BAP, or 1 milligram / liter of NAA + 5 milligrams / liter of GA, and the boxes move toward the lumen. After 2 to 4 weeks, the tissue is transferred to an MS medium without hormones. The sprouts that appear are placed in containers with an MS medium without hormones, or with an MS medium with 0.5 milligrams / liter of NAA. When sufficient root and stem growth occurs, the seedlings are transferred to the soil and placed in a phytotron.

Weeks and collaborators, Plant Physiol. 102: 1077-1084 (1993) Wheat transformation using the method of Weeks et al., Plant Physiol. 202: 1077-1084 (1993), is briefly stated as follows: wheat plants (Tri ticum aestivum L.) are grown, and immature embryos of 0.5 to 1 millimeter in length are cut from plants grown in the greenhouse (from 10 to 18 days after anthesis, depending on the time of year), and placed, with the exposed scutellum side, on a callus maintenance medium containing 1.5 milligrams / liter of 2,4-D. Five days after the initiation in tissue culture, one can see the proliferating callus tissue on the edges of the embryos. At this stage, the embryos are bombarded with gold particles coated with 7 milligrams of pJS20293 and 7 milligrams of pUBIAc.

Bombardment of Particles Before bombardment, 1-millimeter gold particles are coated with DNA from pJS20293 and pUBIAc by the procedure of Daines, Biolistic Systems Newsletter 2: 1-4 (1990). A suspension of the supply of gold particles (Bio-Rad) is suspended at 60 milligrams / milliliter in absolute ethanol. 35 microliters of the suspension are aliquoted into 1.5 milliliter microcentrifuge tubes, washed in sterile distilled water, and resuspended in 25 milliliters of Tris-EDTA containing 25 milligrams of supercoiled plasmid DNA. The following solutions are added in order: 220 milliliters of sterile water, 250 milliliters of 2.5 M CaCl2, and 50 milliliters of 0.1 M spermidine (free base). The microcentrifugal tubes are shaken with a vortex stirrer at 4 ° C for 10 minutes, and centrifuged at 16,000 rpm for 5 minutes. The supernatant is removed, and the button is washed with 600 milliliters of ethanol. The gold granules coated with DNA are resuspended in 36 milliliters of ethanol. For the bombing, they are placed milliliters of DNA-gold suspension in the center of a macroprojectile (aka Carrier Sheet). Approximately 25 embryos are placed in the center of a 15 x 100 millimeter Petri box containing callus maintenance medium solidified with Phytagel at 0.35 percent. After 5 days in culture, vacuum embryo-derived calli are bombarded with gold particles coated with pJS20293, using the helium-driven Dupont Biolistic Supply System, and the disposable components supplied by Bio-Rad. The distance from the stop plate to the target is 13 centimeters, and the force of the rupture disc is 77 kg / cm2. Immediately after the bombardment, the calli are transferred to a selection means MS containing the appropriate amount of selective agent, as can be determined by one skilled in the art.

Regeneration of Wheat Plants For regeneration, embryogenic calli are transferred to an MS medium containing 0.5 milligrams / liter of dicamba, as described by Hunsinger and Schauz, Plant Breeding 58: 119-123 (1987). Sprouts derived from callus are transferred to Pyrex culture test tubes containing a rooting medium composed of medium strength MS without hormones. For selection after bombardment, the agar medium in each stage is supplemented with the appropriate amount of selective agent, as may be determined by one skilled in the art. The seedlings are transferred from the rooting medium to the soil mixing pots, and are acclimated to lower humidity at 21 ° C in an environmental chamber. After 2 weeks, the plants are transferred to the greenhouse. These primary transgenic regenerants are called T0 plants.

Analysis of Transgenic Plants Transgenic tissues and plants are analyzed using Southern and Northern techniques to demonstrate the presence of the peroxidase coding sequence and RNA, respectively. Wheat plants that were shown to contain the peroxidase coding sequence by means of Southern analysis are evaluated to determine their insecticidal activity against Pseudaletia unipunctata, worm worm Spodoptera frugiperda, worm warrior of autumn; Elasmopalpus lignosellus, minor corn stem borer; Agrotis orthogonia, worm of pale western cut; Oulema melanopus, cereal leaf beetle; Hypera punctata, cloverleaf weevil; Diabrotica undec impune tata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, green bug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, red-footed grasshopper; Melanoplus differentialis, differential grasshopper; Melanoplus sanguinipes, migratory skipjack; Mayetiola destroyer Hessian fly; If todiplosis mosellana, wheat midge; Meromyza americana, wheat stem moth; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco trips; Cephus cinctus, wheat stem sawfly; or Aceria tulipae, wheat curly mite, employing well-known techniques in this field. Transgenic wheat plants that have insecticidal properties undergo field trials.

Example 3: Transgenic Sorghum Plants Comprising a Peroxidase Coding Sequence pJS20293 (Figure 1) and pUBIAc (Figure 2) are used to transform sorghum, using the method of Casas et al., Proc. Nati Acad. Sci. USA 90: 11212-11216 (December 1993), as briefly described as follows: Microprojectile bombardment Experiments are conducted with the Biolistics PDS 1000 / He system (Sanford et al., Technique J. Methods Cell Mol. Biol 3: 3-16 (1991)) using tungsten microprojectiles (M-25, 1.7 millimeters in diameter, Dupont No. 75056), or gold (1.5 to 3.0 millimeters in diameter, Aldrich No. 32,658-5). Gold particles (3 milligrams) or tungsten (0.75 milligrams) previously washed in ethanol, in an aqueous suspension (50 milliliters), are coated with 5 to 10 milligrams of the Plasmid DNA, as described by the manufacturer (Bio-Rad).

The bombardment pressures and the distances from the launch plate are determined experimentally. Immature zygotic embryos are bombarded, from 10 a per plastic Petri box (15 x 60 millimeters) between 24 and 72 hours after the culture on the medium. The embryos are transferred to filter papers (4.5 centimeters in diameter) that are previously moistened but do not saturate with the liquid medium. The filter papers work to absorb water from the surface of the embryos, and the embryos are left for 2 to 3 hours on the papers before the bombardment.

Immediately after the bombardment, the immature embryos are removed from the papers and transferred to a semi-solid medium.

Regeneration of Transgenic Plant and Evaluation of its Resistance to Insects The procedures for the selection and maintenance of embryogenic tissue and the formation of shoots and roots from organized structures are as described (Cai S Butler, Plant Cell Tissue Organ Cul t 20: 101-110 (1990)).

The appropriate selective agent is used, as is known to one skilled in the art. Transgenic tissues and plants are analyzed using Southern and Northern techniques to demonstrate the presence of the peroxidase coding sequence and RNA, respectively. The PAT activity is evaluated in leaf and leaf extracts according to DeBlock and collaborators EMBO J. 6: 2513-2518 (1987). The sorghum plants that were shown to contain the peroxidase coding sequence by Southern analysis are evaluated for their insecticidal activity against Chilo partellus, sorghum borer; Spodoptera frugiperda, autumn worm; Helicoverpa zea, corn cob worm; Elasmopalpus lignosellus, minor corn stem borer; Fel tia subterr anean, granulated cutworm; Phyllophaga crini ta, white worm; Eleodes, Conoderus, and Aeolus spp. , wire worms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, corn mincer; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinche; Contarinia sorghicola, sorghum gnat; Tetranychus cinnabarinus, carmine spider mite; or Tetranychus urticae, two-spotted spider mite, using techniques well known in the field. Transgenic sorghum plants that have insecticidal properties are subjected to field trials.

Example 4: Transgenic Rice Plants Comprising a Peroxidase Coding Sequence pJS20293 (Figure 1) and pUBIAc (Figure 2) are used to transform rice, using the methods of Shimamoto et al., Nature 338: 214-211 (1989) (rice) ); Christou et al., Biotechnology 9: 957-962 (1991) (rice); Datta et al., Bio / Technology 8: 736-740 (1990) (rice) and / or Hayashimoto et al., Plant Physiol. 53: 857-863 (1990) (rice). The transformation of rice using the method of Christou et al., Biotechnology 9: 957-962 (1991), is briefly stipulated as follows: Preparation of DNA Gold particles coated with DNA are prepared by mixing the gold particles (10 milligrams) with a DNA solution (20 milligrams) in 100 milliliters of regulator (150 mM sodium chloride, 10 mM Tris-HCl, pH 8.0), and vortex gently for 5 to 10 seconds. Spermidine (100 milliliters of a 0.1M solution) and 100 milliliters of a 25 percent PEG solution (Molecular Weight: 1300-1600) are added with vortexing, followed by the dropwise addition of 100 milliliters of calcium chloride ( 2.5 M). The mixture is allowed to stand at room temperature for 10 minutes, and then centrifuged in a microcentrifuge. The supernatant is removed, and the gold precipitated with the DNA complex is resuspended in 10 milliliters of 100 percent ethanol. The resulting suspension is then coated on an 18 x carrier sheet 18 millimeters, at a concentration of 163 milliliters per carrier sheet, or a calculated concentration of 0.05 milligrams / square centimeter.

Isolation of Immature Embryos and Preparation for Particle Bombing Immature rice embryos from 12 to 15 days old are harvested from expanded panicles and sterilized with 2 percent sodium hypochlorite for 5 minutes. Subsequently, they are rinsed repeatedly with sterile distilled water, and the glumes are removed under a dissecting microscope. The embryos are then removed aseptically and coated on a water-agar plate with the adaxial side in contact with the medium.

Bombardment of Particles The carrier sheet carrying the pearls is loaded onto a particle accelerator, which uses the discharge of a high-voltage capacitor through a small droplet of water as the driving force. A 100 mesh retention screen is placed between the sheet and the target tissue suspended above the machine. Then the assembly is evacuated up to 500 mm Hg to reduce the aerodynamic drag. 10 to 16 Kv are discharged from a 2 mF capacitor through a 10 milliliter water droplet inside the expansion chamber. In this way the sheet is blown against the retention screen, allowing the gold particles to continue forward to impact the target tissue suspended above the screen. The immature directed embryos are placed on a water-agar plate, such that, when the plate is inserted on the mesh, the embryonic scutellar region would remain in the direct path of the accelerated particles.

Plant Regeneration Following the bombardment of particles, the embryos are placed on an MS or CC medium supplemented with 2,4-D at 0.5 or 2 milligrams / liter, and embryogenic callus and seedlings are recovered as described (Hartke, S and Lara, H., Genet &Breed 43: 205-214 (1989), Datta SK et al., Plant Sci. 67: 85-88 (1990)).

Recovery of Callus and Transformed Embryogenic Plants Callus and transformed plants are recovered under both selective and non-selective conditions. In experiments where selection is incorporated into the transformation / regeneration protocol, the appropriate amount of selective agent is used, as is known to those skilled in the art. In this way the putative transformants are identified, subjected to molecular and genetic analysis to confirm the stable integration and inheritance of the introduced gene.

Analysis of Transgenic Plants Transgenic tissues and plants are analyzed using Southern and Northern techniques to demonstrate the presence of the peroxidase coding sequence and RNA, respectively. The PAT activity is evaluated in callus and leaf extracts according to DeBlock and collaborators, EMBO J. 6"; 2513-2518 (1987) .The rice plants that were shown to contain the peroxidase coding sequence by Southern analysis are evaluated. determine its insecticidal activity against Diatraea saccharalis, sugarcane borer, Spodoptera frugiperda, autumn worm, Helicoverpa zea, corn cob worm, Colaspis brunnea, grape colaspis, Lissorhoptrus oryzophilus, rice water weevil, Sitophilus oryzae, rice weevil, Nephotettix nigropictus, rice grasshopper, Blissus leucopterus leucopterus, chinche; or Acrosternum hilare, green mincer, using methods well known in the art.

Example 5: Transgenic Oats Plants Comprising a Peroxidase Coding Sequence pJS20293 (Figure 1) and pUBIAc (Figure 2) are used to transform oats, using the methods of Somers et al., Bio / Technology 10: 1589-1594 (December 1992) (oats) and / or Torbert et al., Plant Cell Reports 14: 635-640 (1995) (oats). Oat processing using the method of Somers et al., Bio / Technology 20: 1589-1594 (December 1992) is briefly stipulated as follows: Cell Cultures Immature embryos from oat lines derived from GAF-30 / Park are used to initiate the callus (Riñes H.W. &Luke, H.H., Theor.Appl. Genet 71: 16-21 (1985)). Fragile embryogenic callus lines are visually selected (Bregitzer, P., et al., Crop Scí 29: 198 -803 (1989)) and subcultured every two weeks on a MS2D medium solidified with 0.2% Gelrite containing MS salts. (Murashige, T. &Skoog, F., Physiol. Plant 15: 413-491 (1962)) with 150 milligrams / liter of asparagine, 0.5 milligrams / liter of thiamine-HCl, 20 grams / liter of sucrose, and 2.0 milligrams / liter of 2,4-D, pH of 5.8. Suspension cultures are started by placing approximately 1 gram of brittle embryogenic callus in 35 milliliters of liquid MS2D medium. The cultures in suspension are selected to determine the presence of small aggregates of cytoplasmically dense yellow cells, and are subcultured every week.

Preparation of DNA Coated Particles Tungsten particles are coated with DNA from pJS20293 (Figure 1), and pUBIAc (Figure 2), using procedures similar to that described by Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) . The previously washed Tungsten particles (1.25 milligrams) are resuspended in 250 milliliters of sterile water in an 1.5 milliliter Eppendorf tube. An aliquot of 25 milliliters of 1 milligram / milliliter of each DNA, 250 milliliters of 2.5 M CaCl2, and 50 milliliters of 0.1 M spermidine (free base) is added to the Eppendorf tube in that order. The mixture is vortexed using a Vortex Genie 2 (Scientific Industries, Inc.) at full speed for 1 minute, placed on ice for 5 to 10 minutes, and centrifuged at 14,000 rpm for 1 minute in an Eppendorf 5415 centrifuge. After centrifugation, 550 milliliters of supernatant are pipetted and discarded. The tungsten particles coated with DNA are resuspended by pipetting them up and down several times, and 1 milliliter of the particle suspension with DNA is loaded onto the macrocarrier of the particle delivery system.

DNA supply The suspension culture cells are rinsed three times with an MS2D medium that lacks asparagine before bombardment. 3 to 5 days after subculturing, the suspension culture cells are harvested by vacuum filtration on an MF Millipore AP support cushion of 10 4.7 centimeters in diameter (Millipore Corp.) to form a uniformly spread thin layer of approximately 0.5 grams of tissue culture cells in fresh weight. Then the beads that support the cells are transferred to Petri dishes of 60 x 20 millimeters. For the bombing of the callus, fragile embryogenic callus (0.5 grams) of 2 weeks of age is uniformly spread over the Millipore support cushions previously moistened with 2 milliliters of MS2D less asparagine medium in Petri dishes of 60 x 20 millimeters. Petri dishes containing suspension or callus samples are placed (Gordon-Kamm, et al., Plant Cell 2: 603-618 (1990)) at a distance of 5 centimeters from the stop plate, and bombarded with the System Supply of Biolistic PDS-1000 particles (gun powder) (DuPont Co.).

Selection of Transformants Following the bombardment, the cells are washed from each Millipore support pad with 5 milliliters of liquid MS2D medium lacking asparagine, in a 60 x 20 millimeter Petri dish, which is then sealed with parafilm, and incubated 21 ° C to 23 ° C in the dark. After 5 days of incubation in the liquid medium, the bombarded cells are placed in a thin layer on 7.0-inch diameter Whatman No. 1 filter paper discs superimposed on a MS2D selection medium solidified with Gelrite lacking asparagine, and containing 3 milligrams / liter of phosphinothricin (PPT) (Crescent Chemical Co. Inc.). The cells of a bombardment are normally distributed over two or more filter papers, depending on the cell density. The filter papers with superimposed cells are transferred to a fresh selection medium at intervals of 2 to 3 weeks. The PPT-resistant colonies begin to appear 7 to 8 weeks after bombardment, and are directly subcultured on the fresh selection medium without filter paper every two to three weeks thereafter.

Regeneration of the Plant Tissue-resistant tissue cultures are placed on a regeneration medium of N + B oats plants (Bregitzer, P., and collaborators Crop Sci 29: 198 -803 (1989)) (Sales MS (Murashige, T . &Skoog, F., Physiol. Plant 25: 473-497 (1962)), 2 milligrams / liter of naphthaleneacetic acid, 0.2 milliliters / gram of benzylaminopurine, containing 3 milligrams / liter of PPT. After 2 to 6 weeks, the callus shoots are removed, and transferred to the MS medium without hormones, but containing 3 milligrams / liter of PPT for root formation. The rooted plants are transferred to a mix of potting soil, and are grown until mature in growth chambers.

Analysis of Transgenic Plants Transgenic tissues and plants are analyzed using Southern and Northern techniques to demonstrate the presence of the peroxidase coding sequence and RNA, respectively. The PAT activity is evaluated in callus and leaf extracts according to DeBlock et al., EMBO J. 6.-2513-2518 (1987). Oats plants that were shown to contain the peroxidase coding sequence by Southern analysis are evaluated for their insecticidal activity, using methods well known in the art.

Example 6: Transgenic Barley Plants Comprising a Peroxidase Coding Sequence pJS20293 (Figure 1) and pUBIAc (Figure 2) are used to transform barley, using the methods of Wan et al., Plant Physiol. 104: 37-48 (1994) and / or Jahne et al., Theor. Appl. Genet 89: 525-533 (1994). The transformation of barley using the method of Wan et al., Plant Physiol. 104: 31-48 (1994) is briefly stipulated as follows: Plant Materials Cultivate plants of spring barley (Hordeum vulgare L.) Golden Promise in cultivation chambers under a period of 16 hours of light / 8 hours of darkness at 12 ° C, and with a humidity of 60 to 80 percent (Hunter, CP, Plant Regeneration from Microspheres of Barley, Hordeum vulgare, PhD Thesis, Wye College, University of London, Ashford, Kent (1988)). The light levels at the top height are approximately 350 to 400 mE. The seeds of a winter crop, Igri, are germinated in the soil in the cultivation chamber under the same conditions. When they are approximately 10 centimeters tall, the seedlings are vernalized for 8 weeks under a period of 10 hours of light (10-15 mE) / 14 hours of darkness at 4 ° C (Hunter, CP, Plant Regeneration from Microspores of Barley, Hordeum vulgare, PhD Thesis, Wye College, University of London, Ashford, Kent (1988)). After vernalization, they are cultivated under the same regime as Golden Promise plants. All plants are fertilized with Osmocote (Sierra, 17-6-12 lower) at the time of planting, and then biweekly with Verdi at 0.02 percent (Peter's, 20-20-20).

Immature Embryos and Callus Derived from Immature Embryos Spikes of the Golden Promise variety with immature embryos of a size of approximately 1.5 to 2.5 millimeters, are superficially sterilized in 20 percent (volume / volume) bleach (5.25 percent sodium hypochlorite ) for 5 minutes, rinse briefly 3 times, and wash for 5 minutes with sterile water. The immature embryos are dissected from the young caryopses, and left intact, or bisected longitudinally. For the induction of the callus for bombardment, the embryos (intact or bisected) are placed with the scutellum side down on a callus induction medium, which is a medium of Murashige and Skoog (Murashige, T. &Skoog, F., Physiol Plant 15: 413-491 (1962)) supplemented with 30 grams / liter of maltose, 1.0 milligrams / liter of thiamine-HCl, 0.25 grams / liter of myo-inositol, 1.0 gram / liter of casein hydrolyzate, 0.69 grams / liter of Pro, and 2.5 milligrams / liter of dicamba, solidified by 3.5 grams / liter of Gelrite (Scott, Carson, CA) or Phytagel (Sigma). The embryos are incubated at 25 ° C in the dark, and the embryogenic callus is selected for bombardment after two weeks.

Cultivation of Anther and MicroSpore Derived Embryos (MDEs) The ears, wrapped by the broad leaves, are harvested from plants of the Igri variety, when the microspores are in the uninucleate medium until the first binucleate stages, and are sterilized in surface briefly with 70 percent ethanol. The anthers are dissected from the spikes, and 60 anthers are placed in each Petri dish (35 x 10 millimeters) with 3 milliliters of 0.3 M mannitol. The Petri dishes are sealed with parafilm, and incubated at 25 ° C in the darkness for 3 or 4 days. The anthers are subsequently transferred to Petri dishes with 3 milliliters of Hunter's liquid FHG medium (a modified Murashige and Skoog medium, with lower NH4N03, and high Gln, Kasha, K. et al., "Haploids in Cereal Improvement: Anther and Microspore Culture, "JP Gustafson, ed, Gene Manipulation in Plant Improvement II, Plenum Press, NY (1990), pages 213-235), without Ficoll-400, and supplemented with 1 milligram / liter of IAA, and 0.2 milligrams / liter of kinetin (designated as FHG +), and incubated as described. Embryos derived from microspores are visible after approximately 2 or 3 weeks, and are used for bombardment after approximately 4 weeks.

Preparation of Microprojection and Bombardment Plates One day before bombardment, IEs (1.5-2.5 millimeters) of young Golden Promise caryopses are cut longitudinally in half, and placed in three different orientations (the Squeeze it up, the side of the scutellum down, or the surface cut upwards) on a medium of callus induction in the center of the Petri dishes (100 x 15 millimeters). For the bombardment of the callus, approximately 0.5 grams of embryogenic callus are cut from cultured IEs, in small pieces (approximately 2 millimeters), and placed in the center of a Petri dish (100 x 15 millimeters) containing medium callus induction. Embryos derived from microspores are harvested from the anther culture dishes using a Pasteur pipette, and are distributed evenly in Petri dishes (100 x 15 millimeters) on a piece of 5 cm Whatman No. 3 filter paper supported by two filters. 7 centimeters Before the bombardment, remove excess medium from the filters. The plasmid DNA is adsorbed on the gold particles (1.0 mm, Dupont, Wilmington, DE) as described above (Daines, RJ, Biolistic Delivery Systems Newsletter 1: 1, 4 (1990)) When two plasmids are used, Equal amounts (milligrams) of DNA are mixed from the two plasmids. All target materials are bombarded once, using the DuPont PDS 1000 He Biological Supply System. The target materials are placed approximately 13 centimeters below the microprojectile stop plate; Rupture discs of 77 kg / cm2 are used.

Transformers of Selection IEs and Callo One day after the bombardment, the half embryos and the pieces of callus are transferred individually to a means of induction of callus with 5 milligrams / liter of bialaphos; the half embryos are cultivated with the scutellum side down, regardless of their orientation during the bombardment. The woven remains on the first selection plate for approximately 10 to 14 days. When transferred to the second selection plate (5 milligrams / liter of bialaphos), individual embryos that are forming calluses, or pieces of callus, are broken using forceps into several small pieces, and kept separately. During the next two to three selection steps (each approximately 10 to 20 days, to 5 milligrams / liter of bialaphos), pieces of callus showing evidence of more vigorous growth are transferred earlier to the new selection plates , and the fabric is handled in an identical way. All callus tissue that is originally developed from each piece of embryo or callus is defined as a line. Callus lines resistant to bialaphos are maintained by a monthly subculture on a callus induction medium with 5 milligrams / liter of bialaphos.

MDES Following the bombardment, several drops of an FHG + medium are added to the MDEs. After 2 or 3 days, the embryos > 1.5 millimeters are transferred individually to the callus induction medium with 3 or 5 milligrams / liter of bialaphos. The smaller embryos remain on the filters, and are transferred to a selection medium when they are approximately 1.5 millimeters. Filter papers are washed every two or three days by repeated addition and removal of FHG + liquid medium. The MDEs remain on the first selection medium for 10 to 20 days. The MDEs that show evidence of callus formation are transferred to a fresh selection medium with 5 milligrams / liter of bialaphos. During the transfer, each MDE that is forming callus breaks into a few small pieces. Other selections as described in the previous section.

Plant Regeneration and Herbicide Application Plants are regenerated from PAT positive callus lines, transferring the embryogenic callus to a FHG medium with 1 milligram / liter of bialaphos at 23 ° C or 25 ° C under fluorescent lights ( 45-55 mE, 16 h / d). In about 2 weeks, seedlings are observed. The green seedlings, of approximately 2 centimeters, are transferred to Magenta boxes containing seedling culture medium (hormone-free callus induction medium) with 1 milligram / liter of bialaphos.

Before they grow to the top of the box, the seedlings are transferred to 15.24 cm pots containing Supersoil and placed in the greenhouse (16 light period, 15 ° C-18 ° C). The regenerants grow to maturity and self-pollinate. Some of the plants are tested to determine their response to Basta (200 grams / liter PPT, Hoechst AG, Frankfurt, Germany) by spraying with a 0.5 percent (volume / volume) solution plus 0.1 percent Tween 20.

The plants are also regenerated from wild type callus on the medium without bialaphos.

Analysis of Transgenic Plants Transgenic tissues and plants are analyzed using Southern and Northern techniques to demonstrate the presence of the peroxidase coding sequence and RNA, respectively. The barley plants that were shown to contain the peroxidase coding sequence by Southern analysis are evaluated for their insecticidal activity against Ostrinia nubilalis, European corn borer; Agrotis Ípsilon, black-cut worm; Schizaphis graminum, green bug; Blissus leucopterus leucopterus, chinche; Acrosternum hilare, green picador; Euschistus servos, chestnut mincer; Hylemya platura, corn seed moth; Mayetiola destructor, Hessian fly; Thysanoptera, Thysanoptera; or Petrobia latens, brown-white mite, using methods well known in the art.

* * * * * All publications mentioned hereinabove are hereby incorporated by reference in their entirety. Although the above invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of this disclosure, that different changes in form and detail may be made, without departing from the spirit of the invention. true scope of the invention and the appended claims.

Claims (29)

1. CLAIMS 1. A method for controlling insects, which comprises feeding or contacting an insect with an insecticidal amount of cells of transgenic monocotyledonous plants, wherein these plant cells comprise recombinant DNA comprising a coding sequence encoding peroxidase.
2. 2. The method according to claim 1, wherein this method comprises transforming a monocot plant with a DNA sequence comprising a coding sequence encoding peroxidase.
3. 3. The method of claim 1, wherein said DNA sequence comprises a promoter operably linked to the coding sequence.
4. 4. The method of claim 3, wherein said DNA sequence comprises a chimeric gene comprising a promoter operably linked to the coding sequence.
5. 5. The method of claim 1, wherein the peroxidase is an anionic peroxidase.
6. 6. The method of claim 1, wherein the cells are obtained from a fertile transgenic plant. The method of claim 6, wherein the plant is selected from the group consisting of wheat, rice, oats, barley, sorghum, and corn. The method of claim 7, wherein the plant is a corn plant. The method of claim 8, wherein the maize plant is derived from a maize line selected from the group consisting of CG00526, CG00615, and CG00714. The method of claim 1, wherein this plant is transformed using a technique selected from the group consisting of bombardment of particles, electroporation, and treatment with polyethylene glycol. The method of claim 1, wherein the DNA sequence further comprises a selectable or classifiable marker gene. The method of claim 11, wherein the marker gene encodes an enzyme selected from the group consisting of neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2,2-dichloropropionic acid dehalogenase, acetohydroxy acid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, haloaryl-nitrilase, acetyl-coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyltransferase, and β-glucuronidase. The method of claim 1, wherein the insect originates from an order selected from the group consisting of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Malophagous, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermápteros, Isoptera, Anoplura, Sifonápteros, and Trichopter The method of claim 13, wherein the insect is selected from the group consisting of European corn borer, corn stem borer, black cut worm, corn cob worm, autumn worm, borer of southwestern corn, minor corn stem borer, sugarcane borer, western corn rootworm, northern corn rootworm, southern corn rootworm, wireworms, northern masked bumblebee, Southern masked bumblebee, Japanese beetle, corn beetle, corn chopper, corn leaf aphid, corn root aphid, chinch, red-legged grasshopper, migratory grasshopper, corn seed moth, corn-stained leaf cutter , Thysanoptera of grass, thief ant, and two-spotted spider mite. 15. A fertile transgenic monocot plant comprising recombinant DNA comprising a coding sequence that encodes peroxidase. 16. The plant of claim 15, wherein the expression of peroxidase confers on the monocotyledonous plant a phenotypic trait selected from the group consisting of insect resistance and altered lignin production. 17. The plant of claim 16, wherein the phenotypic trait is resistance to insects. 18. The plant of claim 15, wherein the peroxidase coding sequence is an anionic peroxidase gene. The plant of claim 15, wherein this plant is selected from the group consisting of wheat, rice, oats, barley, sorghum, and corn. The plant of claim 19, wherein this plant is a corn plant. The plant of claim 20, wherein the corn plant is of a maize line selected from the group consisting of CG00526, CG00615, and CG00714. 22. The plant of claim 15, wherein the DNA sequence further comprises a selectable or classifiable marker gene. The plant of claim 22, wherein the marker gene encodes an enzyme selected from the group consisting of neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2,2-dichloropropionic acid dehalogenase, Acetohydroxy acid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, haloarylnitrilasa, acetyl-coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyltransferase, and β-glucuronidase. 24. A cell, tissue, or seed of a transgenic plant, obtained from the plant according to claim 15, comprising recombinant DNA comprising a coding sequence that encodes peroxidase, wherein the expression of peroxidase confers on the cell, tissue or seed, a phenotypic trait. 25. A transgenic offspring of the plant according to claim 15, comprising recombinant DNA comprising a coding sequence encoding peroxidase, wherein the expression of peroxidase confers on the plant a phenotypic trait. 26. A cell, tissue, or seed of a transgenic plant obtained from the descendant according to claim 25, comprising recombinant DNA comprising a coding sequence encoding peroxidase, wherein the expression of peroxidase confers to the cell, tissue or seed, a phenotypic trait. 27. The use of a recombinant DNA encoding peroxidase to confer insect resistance to cells of monocotyledonous plants. 28. The use of a monocotyledonous plant comprising recombinant DNA encoding peroxidase to control damage to the plant caused by insect attack. 29. An agricultural method, which comprises seeding or harvesting a transgenic plant material, or of plants comprising recombinant DNA comprising a coding sequence that encodes peroxidase.