MXPA97006263A - Methods to maintain sterility in plan - Google Patents

Abstract

A method for maintaining sterility in plants is described. The method comprises the genetic transformation of parental plant lines with a sterility gene that is genetically linked to a resistance gene, which confers resistance to a selective agent, increases the transgenic paternal line and coat the seed of the increased transgenic paternal line with a composition comprising the selective agent to which the resistance gene confers resistance. The resistance gene may be under the control of an inducible gene, or, alternatively, under the control of specific constitutive or tissue promoters, including semi-specific promoters.

Description

METHODS FOR MAINTAINING STERILITY IN PLANTS DESCRIPTION OF THE INVENTION The present invention relates to a method for maintaining sterility genes in plants, and to plants produced using the method. The objective of the reproduction of plants, is to combine, an individual variety, or several desirable hybrid characteristics of the parent lines. For field grains, these characteristics may include resistance to diseases and insects, tolerance to heat and dryness, reduction of time for grain maturity, increased production and improved agricultural quality. With the mechanical harvesting of many grains, the uniformity of plant characteristics such as germination and establishment, speed of growth, maturity, and size of the fruit, is important. The field grains are reproduced through techniques that have the advantage of the plant pollination method. A plant is self-pollinated if the pollen of a flower is transferred to it, or other flowers of the rt-is: plant. A plant is pollinated by crossing if the pollen comes from a flower of a different plant. In Brassica, the plant is normally self-sterile and can only be cross-pollinated. In self-pollination species, such as soybean and cotton, the male and female organs are anatomically juxtaposed. During natural pollination, the male reproductive organs of a given flower pollinate the female reproductive organs of the same flower. Corn plants (Zea mays L.) present a unique situation in which they can be reproduced through techniques of both self-pollination and crossing pollination. The corn has male flowers, located on the spike and female flowers, located on the cob, on the same plant. It can be self-pollinated or cross-pollinated. Natural pollination occurs in corn when the wind blows the pollen from the ears towards the stigmas that come from the upper parts of the incipient ears. A reliable method to control fertility in plants could offer the opportunity to breed improved plants and produce hybrids. In particular, the control of male fertility is significant in relation to the commercial production of hybrids. This is especially true for the development of grain species sold only as hybrids, for example, sorghum and corn, both of which are historically based on some classification of male sterility system. In addition, such a method could be useful in grains such as soybean, sunflower, canola, wheat and others, which in the past, have not been receptive to hybridization.

The production of corn hybrids requires the development of essentially homozygous innate lines, the crossing of these lines, and the evaluation of the crosses. Reproduction and recurrent selection are two of the reproduction methods used to develop innate population lines. Breeding programs combine desirable characteristics of two or more of the innate lines or several broad-based sources in breeding groups, from which new innate lines are developed through fertilization and selection of desired phenotypes. A variety of hybrid corn is the crossing of two innate lines, each of which may have one or more desirable characteristics that lack the other or which complement the other. The new innate lines are crossed with other innate lines and the hybrids of these crosses are evaluated to determine which have commercial potential. The progeny of the first generation is designated F. In the development of hybrids only F ^ hybrid plants are sought. The F1 hybrid is more vigorous than its innate kinships. This hybrid vigor, or heterosis, can be manifested in several ways, including increased vegetative growth and increased yield. These manifestations of heterosis are particularly significant from a commercial point of view; Increased yield is particularly important, both for corn sowing companies and farmers, and high yield hybrid corn lines have become the commercial products of choice in the corn seed industry. The hybrid maize seed is typically produced by a male sterility system incorporating manual depletion. The alternating strips of two innate varieties of corn are planted in a field, and the spikes that carry the pollen of one of the innate (female) are removed. With sufficient isolation of the sources of foreign corn pollen, the ears of the in-line deconduced lines will be fertilized only with pollen from the other inbred line (male), and the resulting seed is, therefore, hybrid, and will form hybrid plants. Unfortunately, although the manual de-dropping procedure is very reliable, it is also a very laborious procedure, which must be carried out within a specific window of critical time, and if there is a reduction or non-availability of labor, the typical window for the dependonation can be lost. In addition, a female plant will occasionally be blown by a storm and will escape depletion. In addition, environmental factors can cause the plants to produce secondary spikes after the manual depletion is completed. Or, the one that performs the dependonation completely will not eliminate the spike of the plant, or sprouts can be formed under the female plants, or the spikes can be detached while still in the whorl. In such a case, such female plants will successfully shed the pollen and some female plants will self-pollinate. This will result in a seed of the innate female line that is harvested together with the hybrid seed, which is intended to be produced. This is a significant disadvantage, since innate lines generally do not pretend to be used for commercial sale, since they do not have the commercial performance of hybrids, and innate lines typically are not made publicly available unless they are legally protected. Alternatively, the female innate lines can be mechanically depleted. The mechanical dependonation is approximately as reliable as the manual dependonación, but it is faster and less expensive. However, most despeding machines produce more damage to the plants than manual de-discharge, and a final cleaning after mechanical de-discharge always requires a manual de-discharge in any case. In this way, no form of depletion is currently completely satisfactory, and there continues to be a need for alternatives, which further reduce production costs and eliminate self-pollination in the production of hybrid seeds. The procedure of laborious dependonation can be avoided using sterile intact male cytoplasmic lines (CMS). Plants of an intact CMS line are sterile males, as a result of factors that result from geno to cytoplasmic, opposite to nuclear. Thus, this characteristic is inherited through female relatives in corn plants, since only females provide cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another intact line that is not male sterile. The pollen from the intact second line may or may not contribute genes that make fertile males to hybrid plants. Usually, the normal maize seed and the seed produced by CMS of the same hybrid must be mixed to ensure that adequate pollen loads are available for fertilization when the hybrid plants grow. There may also be other disadvantages for CMS. One is a historically observed association of a specific variant of CMS with susceptibility to certain grain diseases. This problem has led to the virtual abandonment of the use of that particular variant of CMS to produce hybrid corn, although other CMS systems are still in use.

Another form of sterility, a type of male genetic sterility, known as nuclear male sterility, is described in U.S. Patents 4,654,465 and 4,727,219 to Brar, et al. However, this form of male genetic sterility requires the maintenance of multiple mutant genes at separate locations within the genome and requires a complex marker system to track genes and make convenient use of the system. Patterson also describes a male nuclear sterility system that incorporates chromosomal translocations, which are effective, but complicated. See United States Patents Nos. 3,861,709 and 3,710,511. Many other attempts have been made to improve these disadvantages. For example, Fabijanski, et al., Developed several methods to induce male sterility in plants (see EPO 89 / 3010153.8, publication No. 329,308, and PCT application PCT / CA90 / 00037, published as WO 90/08828). One method includes supplying the plant with a gene encoding a cytotoxic substance associated with a promoter specific for the male tissue. Another method involves an antisense system in which a gene critical to fertility is identified and a gene, which is antisense to the fertility gene, is inserted into the plant. Mariani et al. Also describe various sequences of the cytotoxin coding gene, together with male tissue-specific promoters and mentions of an antisense system. See EP 89 / 401,194. Still other systems use "repressor" genes which inhibit the expression of another critical male fertility gene. PCT / GB90 / 00102, published as WO 90/08829. Other aspects of work with male sterility systems are related to the identification of genes that impact male fertility. Such a gene can be used in a variety of systems to control male fertility. Previously, a male fertility gene in Arabidopis thaliana was identified through male-sterile mutants induced by transposon, and the gene was then cloned. Aarts, et al., "Transposon Tagging of a Male Sterility Gene in Arabidopsis", Nature, 363: 715-717 (June 24, 1993). Another important male fertility gene is disclosed in the pending United States application Serial No. 08 / 013,739, which is a continuation of part of the pending United States application Serial No. 07 / 537,183. See also Albertsen et al. , Am. J. Botany 80: 16 (1983). Without considering the sterilized system to achieve male sterility, as noted above, male sterility is of critical importance in the production of commercial hybrids. The presence of unsterilized males in a commercial production field can have disastrous consequences, since indirect crosses and / or self-pollinated seeds will be presented, and consequently "contaminants" (in this context, seed that has a genetic development different from that of the hybrid which is intended to be produced), will be present. Once the transgenic male sterile paternal lines have been produced, it becomes crucial to develop a strategy to maintain male sterility genes and eliminate male fertility in the inbred progeny of maize. This is essential for use in commercial seed production. For example, a strategy could be the union of the male sterility gene with the herbicide resistance gene of Bar and maintain its characteristic as a heterozygous through crossing. Crossed progeny of this must segregate 1 sterile: 1 fertile. In this way, fertile plants can be eliminated through the application by sprinkling of an appropriate herbicide: BastaMR for example. Several limitations are associated with this aspect. Spraying herbicides is itself a potentially dangerous practice, and the cost of specialized equipment required for spraying is usually prohibitive. Also, some herbicides can have particularly devastating effects on the health of surviving plants, and in some cases it can have long-term potential effects. For example, it is known that glyphosate can be transferred and stored in the meristematic tissue, and this can negatively affect fertility. However, a significant limitation of this aspect is the displacement of the herbicide, which annihilates the non-transgenic male parent in the production of hybrid seed. However, it will be recognized that these are problems of spraying or application, or treatment problems of the entire plant, of the same herbicidal problems. An alternative method for the application of herbicide that prevents displacement, should solve this problem. The coating of the seed for protection against fungi and for the maintenance of mechanical integrity has been described in U.S. Patent No. 4,438,593 issued to McNew et al. Coating the seed with Captan ™ or phosphinothricin derivatives, using adhesive agents, has also been described in McNew, et al., As well as in U.S. Patent No. 5,145,777 (Goodman, et al.) And the Canadian Patent. No. 1,251,653A. The genetic transformation of plants to produce herbicidal resistance is well known in the science of grains (see for example, U.S. Patent No. 4,975,374). In addition, U.S. Patent No. 5,369,022 discloses a method for improving grain protection from herbicide damage by incorporating genetically imparted resistance to the herbicide in combination with a grain treatment seed with an antidote amount from a chemical insurer for the herbicide. . However, the novel combination of seed coating, genetically induced resistance, and genetic transformation to maintain sterile plants provides a unique method to maintain sterility genes in the progeny which address many of the problems outlined above. The present invention provides a method for maintaining sterility in plants. The method involves the generation of a transgenic parental plant line, which comprises a sterility gene genetically linked to a gene that confers resistance to a selective agent, for example, a herbicide, an antibiotic, a substrate such as 4-methyltriptofan ( 4-mT) or similar. The transgenic paternal plant line is increased through traditional plant breeding methods, and the seed of the increased transgenic paternal plant lines are coated with a composition comprising the selective agent to which the resistance gene confers resistance. Sterility genes useful in the practice of the invention include, for example, the pAN :: Tox gene, the dam ethylase gene, the ACC synthase gene and the like. The invention visualizes that the sterility gene is genetically linked to a gene that confers resistance to a selective agent, for example a herbicide or an antibiotic, or some other suitable agent, for example, a toxic substrate, such as 4-mT. The resistance gene may be under the control of a constitutive promoter or alternatively it may be under the control of a seed-specific promoter, or alternatively it may be under the control of an inducible gene, - for example, one induced by a hormone, or by another chemical substance without hormone, for example, a protein without a hormone or chemical "insurer", or some other form of chemical ligand. According to this embodiment of the method of the invention, the seeds of the parental line are coated with a mixture comprising an effective amount, both of the selective agent to which genetic resistance has been conferred, as well as the chemical that induces resistance. In a further alternative embodiment, the resistance gene may be under the control of a seed-specific promoter. A particular advantage of these embodiments refers to the fact that the expression of the resistance gene can be limited to the stages of seed development. These modalities, therefore, have particular advantages to limit or restrict the expression of genes, which are subject to governmental regulations. Specifically, these embodiments of the invention, in particular the modality that refers to the use of a seed-specific promoter, have the advantage of restricting the expression of the seed resistance gene and avoiding potential regulatory emissions associated with grain strength. , such as sunflower, sorghum and canola, grains where resistance can be crossed to wild species. These modalities provide the additional advantage of avoiding the need to sell seeds, which will give rise to plants that can putatively express resistance genes in the whole plant through the life of the plants. An object of the method of the invention is to provide a system by which the application of a selective agent, a herbicide, for example, to the seeds of plants that will result in the death of a wild-type and surviving plant is presented. of resistant plants, so that spraying or other forms of general environmental application of the whole plant is avoided, and in this way, the death of the wild-type male fertile plant and the survival of resistant plants can be achieved early, and in certain selected modalities, stages of development. A general advantage of the method of the invention is to avoid the need to apply potentially hazardous chemicals on the plants in the field, thereby reducing the amount of such chemicals used in and introduced into the environment. A further advantage of the invention is that the method will encourage the development of hybrids in the self-pollination species, such as soybeans, and in species that have not been susceptible to manual depletion, such as sorghum. In addition, the present invention also visualizes the transgenic seed coated in plants, the growth thereof of sterile male plants, transgenic, mature, the fertilization of mature male sterile plants, and the harvest of the fertilized seed of those plants. The present invention also relates to the seed produced according to the method of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a plasmid map of the plasmid pPHP610. Transgenic parental plant lines can be generated using a number of methods recognized in the art, including, for example, bombardment of icropoyectiles, microinjection, Agrobacterium-mediated methods. , electroporation, and the like. See generally, Glic, and Thompson, Methods in Plant Molecular Biology and Biotechnology, CRC Perss, 1993. As those skilled in the art, they will recognize that the choice of transformation method and conditions will vary depending on the species of plant that will be subjected to the method of the invention.

Any gene construct conferring sterility can be applied in the method of the invention. For example, in a preferred embodiment of the method of the invention, the construction conferring sterility is the pAN :: tox construct. Other genes useful for conferring sterility have been described in the art (see references cited above), and include, for example, dam methylase and ACC synthase. As used herein, the term "sterility gene" refers to a gene, which actively promotes or confers sterility, and may include heterologous genes, such as the Barnase gene, and the dam methylase gene, and the like, or it can be a gene that is antisense to the fertility gene. The present invention also involves the use of a resistance gene, which confers resistance to a selective agent genetically linked to the sterility gene. "Genetically linked" is used in the present to refer to an association, either direct or indirect between the two genes, such that during the transformation, these are released into the plant cell, in the same case of transformation, and are incorporated into the genetic material of the recipient plant, in such a way that during the increase of the paternal seed, the genes maintain their functional association.

The method of the invention relates to genetically binding the sterility gene to a resistance gene, for example, a gene for herbicidal resistance, or for resistance to antibiotic, or genes that alter the metabolism in order to allow the metabolism of toxins. or other selective substrates. The resistance gene confers resistance to a corresponding selective agent: the herbicide to herbicide resistance genes; antibiotic resistance genes to antibiotics, and other chemical resistance genes to their respective chemical agents, for example, the gene for tryptophan decarboxylase, which confers resistance to 4-mT. In one embodiment of the method of the invention, the sterility gene is genetically linked to a gene, which confers herbicidal resistance. Resistance can be conferred to herbicides of various groups, including inhibitors of amino acid synthesis, photosynthesis inhibitors, lipid inhibitors, growth regulators, cell membrane switches, pigment inhibitors, seed growth inhibitors, and the like, and Specific herbicidal examples applicable to the method of the invention include: imidazolinones, sulfonylureas, triazolopyrimidines, glyphosate, sethoxydim, fenoxaprop, glufosinate (for example, bialaphos), triazines, bromoxynil, and the like. See for example, Holt, J.S., Mechanisms and Agronomic Aspects of Herbicide Resistance, Ann. Rev. Plant Physiol. Plant Mol. Biol. 44: 203-29, 1993; Wilmink et al., Selective Agents and Marker Genes for Transformation of Monocotyledonous Plan, Plant Mol. Biol. Rept. 11, 1993; Gunsolus, Chart Your Chemical's Family Tree, Soybean Digest April, 1993. In a specific embodiment of the present invention, the commercial herbicide Basta ™ is used. Suitable genes for use in this embodiment of the method of the invention include, for example, Bar, PAT, aroA, Epsps, corl-1, bxn, and psbA. In an alternative embodiment of the present invention, the sterility gene is genetically linked to a gene that confers antibiotic resistance. Antibiotics useful in the practice of the invention include, for example, aminoglycoside antibiotics, for example, kanamycin, gentamicin, G418, neomycin, paromisin, and hygromycin, and genes useful in practicing this embodiment of the invention, include , for example NPT II, the aphA2 gene of Tn5 from E. coli, and the pht-aphlV gene from E. col i. See Wilmink et al., Op. cit. See also, Bowen, B.A.,? Far-ícers for Plant Gene Transfer, Transgenic Plant. Vol. 1, Engineering and Utilization, 1993; Everett et al., Genetic Engineering of Sun f lower (Helianthus annus L.).

Bio / Technology 5: 1201-1204 (1987); Bidney et al., Plant Mol. Biol. 18: 301-313 (1992).

In a further alternative embodiment of the invention, the sterility gene is linked to a resistance gene that confers resistance to a particular chemical, for example, one that is toxic to the plant. An example could be the gene for tryptophan decarboxylase, which confers 4-mT resistance. • See Goodijn et al., A Chimeric Tryptophan Decarboxylase Gene as a Novel Selectable Marker in Plant Cells, Plant Mol. Biol. 22: 907-912, 1993. See also Bowen, op. cit. In one embodiment of the invention, the resistance gene is under the control of a construction promoter. In an alternative embodiment of the invention, the resistance gene is under the control of a promoter induced by a particular chemical (an inducible promoter). For example, the resistance gene can be induced by a hormone, for example, a protein hormone or a steroid hormone, or through a chemical "insurer". Examples of effective steroid hormones in the practice of the invention, could be estrogens, glucocorticides and the like. See, for example, Gronemeyer, Transcription Activation by Estrogen and Progesterone Receptors, Ann. Rev. Genet. 35: 89-123, 1991. See also Glick and Thompson (cited above), in particular Graber and Crosby, Vectors for Plant Transformation, a pp. 89-119, for a discussion of inducible promoters. For a discussion of other chemical agents, see Hershey and Stoner, Isolation and Characterization of a cDNA Clones for RNA Species Induced by Substi tuted Benzene-Sulfonamides in Corn. Plant Mol. Biol. 17: 679-690. See also U.S. Patent No. 5,364,780 for the discussion of "insurers". In these latter alternative embodiments, it will be obvious to those skilled in the art that the seeds may be coated with mixtures comprising the specific chemical agent to which it responds to the inducible gene, together with the particular selective agent. Alternatively, specific tissue, or more particularly seed specific expression, may be genetically provided. Glick, and Thompson, op. cit. Once suitable transgenic parental lines have been produced, the method of the present invention involves the increase of parental lines, using methods well known in the art of plant breeding. Once the parental lines have been sufficiently increased, the seed of the increased line can be collected by familiar means, and recognized by those skilled in the art of plant breeding. The seed collected from the increased transgenic parent lines can be coated in any suitable manner. For example, in a specific embodiment of the invention, an equal volume of a working solution of active ingredient at 10% Captan ™ - (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide [active ingredient], dipropylene glycol [adhesive ], and polyox (WSRN10) [adhesive] (final concentrations of 42.5% Captan ™, 54.5% dipropylene glycol, and 3% polyox) are mixed with a commercial formulation of Basta ™ (20% phosphinothricin [DL-homoalanin-4 acid] -yl (methyl) -phosphinic, see Droge et al., Plant 187: 142-151, 1992], active ingredient) at a ratio of 1: 1 (vol-vol.) The mixture can be applied to the seeds in any The coating may proceed in a sequential treatment (Captan ™ followed by selective agent and, if applicable, the inducer) or a simultaneous treatment, although the sequential treatment steps are currently preferred.The alternative formulations will be readily apparent to those skilled in the art. in the For example, a mixture of 1: 1 (vol: vol) of a working solution of Captan ™ and methionine sulfoximine (MSO) can be used. Alternative formulations of various binders and alternative selective agents, can be routinely screened for efficacy, by screening the germination of coated seeds with coating mixtures containing increased concentrations of the binder and / or selective agent, and similar screening procedures can be conveniently performed for coatings containing additional alternative chemical ligands to practice the invention using an inducible system. After the coated seeds have dried, they are planted as is appropriate to the type of plant, and the resistant sterile seed will germinate and grow. Then, the production of a hybrid plant can proceed according to methods well known in the plant breeding technique. The following examples are intended to illustrate the invention, but not to limit its scope. EXAMPLE 1 Effect of Seed Coating on Seed Survival This example establishes the results of experiments performed to determine the effect of seed coating on the survival of seeding on wild type and transgenic seeds. Materials and Methods A seed of the variety of transgenic plants TO and Hi-II of wild type was obtained. Unless otherwise mentioned, grains were shredded for PCR analysis to indicate which selfed progeny TO contained or did not contain the Bar gene. Then, the grains were immersed in a 1: 1 Captan ^ R mixture (working solution). of 10% normal active ingredient used in seed production) and the commercial formulation BastaMR (active ingredient 20%). The similar grain coating was performed, using a 1: 1 mixture of Captan ™ and methionine sulfoximine (20% aqueous solution). Then, the seed was allowed to air dry to a characteristic pink color of Captan ™. Positive Bar grains were planted in a separate greenhouse container from those that were negative to Bar. In a subsequent experiment, the grains were not shredded for PCR, but rather were coated with BastaMR-Captan ™, planted in containers of greenhouse and the foliage material of the surviving plants was collected for PCR analysis. The wild type maize grains were either coated with BastaMR-Captan ™ or left untreated and planted in separate containers. Of 13 grains, all untreated germinated and produced plantings. However, none of the grains treated with BastaMR were able to germinate and develop in sowings. These results suggested that the application of BastaMR to the surface of the seed was a successful method to apply a lethal dose of this herbicide. This method of application of herbicides was then repeated with the transgenic seed. Before treatment with BastaMR or MSO, transgenic IT grains were shredded by PCR to determine which Bar were negative and Bar positive. The results of the second seed coating experiment are listed in Table 1. None of the grains that were negative Bar survived to form a seeding, while 80% and 53% of those positive Bar grains were coated with BastaMR or MSO respectively, and plants were developed. Bar Negative grains were viable and germinated to form roots, but failed to form suckers. The same was evident for the Bar negative grains treated with MSO. The disadvantages of coating the grains with Basta ^ or MSO are as follows: First, it is a quick method, whereby thousands of grains can be treated in a matter of minutes. Secondly, this technology is perfectly set with the current use of Captan ™ as a seed treatment. Third, it mitigates the problems associated with spraying, such as the need for cost experiments, displacement of herbicides, and others mentioned above. Fourth, it is an elegant and novel solution to a significant problem. As you can see in Table 1, only about half of the positive Bar grains were able to survive the MSO treatment compared with 85% of those treated with Basta1 ^. The exact reason for this has not been resolved; without pretending that it is limited by theory, it may be that too much MSO was applied to the surface of the grains and that lower levels could result in a better survival of transformed progeny Positive Bar. TABLE 1 EFFECT OF SEED LINING WITH ENOUGH AND MSO IN THE PLANT SURVIVAL 10 DAYS AFTER PLANTING IN THE GREENHOUSE Chemical product Bar Negative Bar Positive Basta 0/15 * 12/15 MSO 0/15 8/15 * E1 numerator refers to the number of surviving crops. The denominator refers to the number of seeds that were planted. EXAMPLE 2 Herbicide Coating Growth Chamber Test and Field Test A preliminary examination was conducted in the growth chamber to test the expression of the herbicide resistance characteristic on four separate pure ancestors. The second portion of this study was a field feature that was conducted in two locations (Sysart, IA and Johnston, IA) during the 1994 growing season. The field trial was covered under the USDA Environmental Release Permit # 94-056-05N.

Materials and Methods 1) Preliminary Growth Chamber Test The treatments for this preliminary test were a factorial combination of four phenotypes and three herbicide coating regimes. The seeds were obtained from two transgenic populations (35S :: BAR) TI and crossing with several female innate relatives: Innate Lines A, B, C and D. On May 6, individual lots were treated (30 seeds per lot), with 0.08 mi from CaptanMR. Immediately after the sprinkling of the Captan ™, each batch was treated with either 0. 0.12 or 0.60 ml ("zero", "low" and "high") of a glufosinate-containing herbicide solution known as BastaMR (Hoechst) to a 20% concentration of active ingredient. A rotary treater manufactured by Hans-Ulrich Hege ("HEGE 11") was used to apply the Captan ™ and Basta ™ treatments. Each of the three plastic boxes was filled with 2 kg of dry soil in the air. On May 6, three rows (15 grams per row) of an individual genotype planted in each box. The rows represented the regimes of either zero, low or high of BastaMR. The dry floor was covered with paper towels and then 500 ml of tap water was emptied onto the towels. The towels were removed once the water penetrated the ground. Boxes were sealed with caps and then placed in a chamber with controlled temperature, in the dark, set at 22 ° C. On May 17, emergency accounts were registered. - Discolored plantings were classified, either as normal or abnormal based on appearance relative to untreated controls. The tissue of the emerged seeds was harvested and used to generate Southern profile data (See Example 3 below). II. Field Analysis The treatments for the experiment were a factorial combination of four genotypes and six herbicide coating regimes. Each genotype was a complete graph, and a single pile (length 5.33 meters) of each herbicide coating regimen was a subgraph. The entire experiment was arranged as a randomized complete block design with three replicas at each location. Seeds were obtained from two transgenic IT populations (35S :: BAR) crossing with the selected female innate parents of Example 1: Innate Lines A, B, C and D. On May 18, small lots (325 seeds per lot) were treated with 0.86 ml of Captan ™ sludge. The next day, the individual seed lots were treated with either 0., 1.30, 2.60, 3.90, 5.20, or 6.50 ml of BastaMR. HEGE 11 was used to apply the Captan ™ and Basta ™ treatments. One day after the application of BastaMR, all the seeds were coated with talcum powder to prevent the grains from adhering during planting. Conventional culture was used to prepare a bed of fine seed at each location. Double herbicide was incorporated for the control of preplant seed to Dysart. Seeds were planted manually (28 grains per pile) at a depth of 2 to 5 cm using a traditional push planter. The dates of the plantations were May 27 for Johnston and June 1 for Dysart. Pounce ™ was applied to track control on June 3 at Johnston. The weed that emerged during the experiment was removed by hand. Emergency data was recorded for each subgraph on June 7 at Johnston and June 17 at Dysart. During June 21-24 in Johnston, and June 27-28 in Dysart, each plant under experiment was assigned an identification number that was recorded on a small plastic pole placed on the ground immediately adjacent to the plant. At the same time, small leaves of a superior leaf of the plant were eliminated, and later they were placed in a marked plastic bottle. The label on the plastic corresponded to the identification number of the plant in the field. The leaves were stored in a freezer for future PCR analysis. On July 6, all containers in both locations were sprayed with BastaMR, using a backpack pump sprinkler loaded with C02. The application rate of BastaMR was 6.4 pints per acre with a vehicle volume of 9.14 liters of water per acre. Three nozzles directed the herbicide towards the top and both sides of the plants. The coverage was excellent, and the weather conditions were hot and dry. The identification numbers of those plants that were sensitive to BastaMR were registered on July 11 at Johnston and July 12 at Dysart. The sensitivity to BastaMR was obvious; the plants were yellow and had areas of necrotic tissue. The resistant plants appeared normal, except for a limited degree of small spots on some leaves. Without claiming to be limited by theory, this effect was probably caused by some non-active ingredient in the herbicide, and was markedly less severe than the general decline observed in sensitive plants. Then the data was collected, the plants were crushed on the soil surface and then the residues were scarified in the soil. The graphs were verified to ensure that none of the plants were able to recover or release pollen. Results and Discussion In the preliminary study of the growth chamber, the emergence was almost 100% for the grains that were not coated with BastaMR; these plantings appeared normal (Table 2). The application of BastaMR reduced the emergency regime. At the low BastaMR regime, some of the emerged plantings were very delayed and seemed abnormal in relation to the controls. It is postulated that these abnormal plants were secreted untransformed that showed herbicidal damage. The minor abnormal plantings occurred at the high regime, unduly since the non-transformed segregates were successfully annihilated by the herbicide. In retrospect, this test should have been conducted in the light instead of the dark, since the herbicide should show greater phytotoxicity in the light. To summarize the results of the field analysis, it was observed that the treatment of grains with BastaMR completely eliminated the emergence with sensitive BastaMR (Table 3). The control of BastaMR sensitive segregates was obtained even at the lowest BastaMR regime applied to the grains. This conclusion was based on the fact that only the plants that showed susceptibility to the herbicide applied to the leaves were in the control charts that did not have BastaMR in the grains (Table 4). The percentage of reduction that resulted from the treatment of the grains was consistent with the expected segregation ratio for the herbicide resistance characteristic. Based on the results of clear cut, it was decided that it is not necessary to perform any PCR analysis on the discs of the leaves that were collected during the experiment. TABLE 2 Results of the Preliminary Growth Chamber Test, Emergencies of Seed Affected by Coating of Grains by BastaMR Genotype Zero Low High (by means of female) # Emerged Intact A15 (all normal) 5 (normal) 7 (normal) 8 (abnormal) 4 (abnormal) Intact B 14 (all normal) 4 (normal) 7 (normal) 1 (abnormal) 1 (abnormal) Intact C 15 (all normal) 8 (normal) 8 (all 2 (abnormal) normal) Intact D 15 (all normal) 5 (normal) 7 (all 5 (abnormal) normal) TABLE 3 Emergency Percentage of Seed Plants Coated with BastaMR and Captan * 41 * Basta Dose Rate MR Female Location Intact ox 1X 2X 3X 4X 6X Johpßton A 905 10.9 * NO 46.4 * 12.4 33.3 * 4.1 35.7 12.9 41.7114.4 B 97.6 * 2.1 35.7 * 4.1 31.0 * 4.1 31.0 * 2.1 36.1 * 4.1 31.0 * 7.4 C 92.9 * 62 35.7112.9 47.6 * 11.1 51.2162 42.9 * 15.6 381 11.5 D 92.9192 405 ± 12.5 54.9111.5 47.61 .1 9.2192 47.6 11.5 Daart A 92.9 * 92 NO 383136 32,116.2 38115.5 39.3 * 9.4 B 96.4 * 0.0 27.4 * 7.4 42.9 * 36 32.1 * 62 35.7 * 17.9 33315.6 C 90.5 * 7.4 512 * 2.1 39.3 * 7.1 54.6 * 13 J 46.417.1 41.7 14 J 0 95.2 * 2.1 42.9 12.9 «about 4661115 54.8 * 20.6 452 5.5 * The values represent means for three applications ± S.D. TABLE 4 Percentages of Plants That Were Sensitive to a Foliar Application of Basta141 Basta Dose Rate MR Intact Female Location ox 1X 2X 3X 4X «X Jonnajtui A 50.0 * 9.4 * 0.0 0.0 00 00 0.0 B 58.3 * 13.5 EYE 0.0 ao 0.0 EYE C 44.0154 0.0 0.0 0.0 0.0 0? O 42-9 * 9.4 0.0 0.0 0.0 0.0 0.0 Oyaart A 486 * 9.0 ao 0.0 0.0 0.0 ?? B 69.0 * 7.4 ao 0.0 0.0 0.0 0.0 C 63 ** 94 Ofi 0.0 00 0.0 00 OR «2.4 * 4.1 ao 0.0 0.0 0.0 0.0 * The values represent means for three replications ± S.D. EXAMPLE 3 Southern Growth Growth Chamber Analysis For corn populations through microparticle bombardment, with the plasmid pPHP610 (Pioneer Hi-Bred International, Inc.) containing the Bar gene. The seed was coated with only the coating of CaptanMR (BastaMR was not added), or CaptanMR plus 0.12 mi from BastaMR or CaptanMR plus 0.60 mi from BastaMR. The plants germinated and the DNA of the surviving plants was extracted and subjected to Southern analysis. In one form of Southern analysis, the putative transformed DNA was digested with a restriction enzyme, which was predicted to be cut once inside the plasmid. This was called "integration analysis" and EcoRl was chosen for the plasmid pPHP610. In an alternative form of the Southern EcoRl / Notl analysis, it was chosen in order to provide a digestion, where the DNA of the released plasmid could be cut twice, and where the digestion will drop a fragment of a known size that will contain both as the plant transmission unit ("PTU") of which it is possible. The "integration analysis" is designed to theoretically give a hybridization mark sized differently, for each different integration site in the plant genome. The "OCT" analysis must hybridize to a specific fragment size. The presence of fragments of hybridization of different size, could indicate a case of complex integration (for example, stirring, elimination or insertion in the PTU region). The results of the 'Southerns' analyzes were analyzed for the presence of or absence of the Bar gene. The Bar coding region was used in all analyzes, and the template for the probes was the 567bp Bam Hl / Hpal fragment from pPHP610. A negative control was used, 10 μg of Hill (F2 of B73 x A188) untransformed DNA. As a positive control, pPHP610 HI-II plasmid DNA was added equal to 1 copy / genome and 5 copies / genome to 10 g of untransformed Hi-II DNA. The integration analysis showed individual transformation cases through the improved lines, with some more complex integrations in certain lines. These lines appeared to have three separate integration sites containing 3 PTUs, one of which appeared to be intact. Only one plant which did not contain the Bar gene germinated from a seed coated with Basta ^ R, and this was not surprising since this plant was of a grain coated with BastaMR plus CaptanMR, which germinated and developed under suboptimal conditions (in Darkness) .

EXAMPLE 4 Induction of Report Gene Sequence Via Seed Coating Seed derived from crossing transgenic TO corn plants contains a suitable hormone dependent receptor and a hormone response element driving the Bar gene with wild type pollen, They were used as the source material. The resulting seed was coated with the following combination of chemicals. 1) Control not coated 2) Coated only with BastaMR 3) Coated only with hormone 4) Coated with BastaMR and hormone The coated seed was planted in the greenhouse and the emergence was quantified. The anticipated results appeared to be almost 100 percent emergency of the uncoated control seed and the hormone-only coated seed. No emergence of the seed coated only with BastaMR was expected and a 50% emergence of the seed coated with BastaMR and hormone was expected. EXAMPLE 5 Safe Induction of the Report Gene Via the Seed Coating Seed derived by crossing corn plants contains a DNA promoter sequence of IN2-1 or IN2-2 that drives the Bar gene with wild type pollen, were used as source material. The resulting heterozygous seed was coated with the following combination of chemicals. 1) Uncoated control 2) Coated with BastaMR only 3) Coated only with the 2-CBSU 4) Coated with both BastaMR and 2-CBSU BastaMR was applied as described in Example 2, while the 2-CBSU was applied at speeds of an equivalent of 7568 liters / hectare. The coated seed was planted in the greenhouse and the emergence quantified. The anticipated results can be almost 100 percent emergency of the uncoated control seed and the seed coated only with the 2-CBSU insurer. No emergence of the seed coated only with BastaMR was expected, while 50% of the seed coated with both BastaMR and with the 2-CBSU insurer was expected. EXAMPLE 6 Specific Seed Resistance Expression There will be cases where it may be desirable to use the seed coating system on seeds that produce plants that do not express more than the resistance beyond the sowing stage. For example, some species of grain plants may be poor choices for the introduction of resistance, since they are crossed with weed species. Although the seed coat treatment will require that a resistance gene be expressed in the seed and possibly in the seeding and / or roots, it does not require that the resistance gene be expressed in the plant in other stages of development in other tissues, such as in the leaves of young or adult plants. There are promoters that could limit the expression of the seed and / or seeding. These include promoters of genes that are expressed in aleuron under germination, such as the a-amylase gene amy32b of barley (Whittier, RF, Dean, DA, and Rogers, JC 1987, Nucleic Acid Res. 6: 2515-2535); genes that are expressed in seed embryo development until very late in seed maturation, such as the corn glbl globulin gene, (Belanger, F.C. and Kriz, A.L. 1991, Genetics 129: 863-872); and genes that are expressed both in the embryo during seed maturation and in seeding during germination, such as the malate synthase gene MS-a from Brassica napus (Comai, L., Matsudaira, KL, Heupel, RC , Dietrich, RA, and Harada, JJ 1992, Plant Physiol, 98: 53-61), or the Em gene, of wheat, which is active in mature seed embryos and ABA inducible in plantings. (Marcotte, W.R., Jr., Russell, S.H., and Quantrano, R.S. 1989, Plant Cell 1: 969-976).

An active promoter in aleuron seed germination could provide the expression necessary to inactivate the chemical seed coat as the seed enters. In the case that some chemical selective agent is able to penetrate the seed without being inactivated by the product of resistance gene in the aleuron, a promoter of late expression in embryo development during seed maturation could provide a source of resistance gene product in the embryo before germination, so that no atrophy of the embryo can occur due to exposure to the agent selective before it can produce the resistance gene product. The expression of the resistance gene to germinate seeds, may be necessary for protection against the residual selective agent in the soil, as well as any selective agent that is not inactivated by the resistance gene produced in the aleuron. Another option to protect plantings of any residual selective agent in the soil could be a resistance gene activated by a specific root promoter, such as the wheat P0X1 peroxidase gene (Hertig, C, Rebmann, G., Bull, Jr. , Mauch, F., and Dudler, R. 1991, Plant Molec., Biol. 16: 171-174). Since the strategy outlined above can be reasonably achieved through an individual expression pattern or a combination of different patterns of expression of separate resistance transgenes, it is also possible for an individual resistance transgene with a promoter or promoters that confer all Specific characters of the necessary tissue can be developed. This may be a promoter having one or more of the specific features described above, a combination of promoters with different expression patterns, or a hybrid promoter derived from two or more promoters having different individual specific characters as discussed above. Regardless of the particular aspect, the seed coating system can be used on seeds that germinate and survive, but produce plants which do not express greater resistance beyond the sowing stage. EXAMPLE 7 Use of the NPT II Gene The seed is obtained from several transgenic and wild-type plants, for example, maize, sorghum, soybean, canola, sunflower, wheat and other cereal or dicotyledonous plants, which express the NPT II gene. The transgenic plants are identified based on the enzyme or elisa analysis for the expression of the NPT II gene, PCR analysis of the structural gene for NPT II, and / or Southern analysis of NPT II. The individual transgenic and wild-type plants are self-harvested to produce the TO seed for analysis. Typically, the presence or absence of the NPTII gene is confirmed by PCR of seed pieces from individual plants. The seeds are submerged in a combination of a Captan solution (10% active solution) and varying levels of kanamycin in an aqueous solution ranging in concentration from 25 mg / 1 to 1000 mg / 1 with the preferred levels of 100 and 400 Both the vacuum infiltration and the simple coating of the seed are made with seeds of each plant. The seed identified as NPT II positive and those identified as NPT II negative (wild type) are planted and subsequently germination is attempted in a soil mixture under greenhouse conditions. A similar test was performed where the wild-type plant and the known transgenic plants expressing NPT II were pollinated with wild-type plants. The seeds of each of the plants were coated as mentioned above, and the seed was planted under greenhouse conditions. The wild type plants were expected to show either no germination, or growth of albino plants. The seeds of the transgenic plants pollinated through wild-type plants were expected to have segregation ratios consistent with a ratio of 1: 1 where normally half was germinated, and the other half either did not germinate or They were albines. The PCR analysis of the resulting green plants was expected to be positive for the presence of the gene in all cases. In another test, kanamycin was sprayed on the sowings with a known identity at levels ranging from 25 mg / l to 1000 mg / l. Those crops that contain the NPT II gene remain green, while wild-type plants did not show any germination or were either an albino phenotype or lost chlorophyll with the leaves present at the time of spraying. The description of all patents and other publications cited herein are incorporated herein by reference. Although the above invention has been described in detail for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain modifications may be made within the scope of the appended claims.

Claims (30)

1. CLAIMS 1. A method for maintaining sterility in plants, the method is characterized in that it comprises generating a transgenic parental plant line, the transgenic parental plant line comprises a sterility gene genetically linked to a resistance gene, which confers resistance to a selective agent, increasing the transgenic parental plant line, and coating the seed of the transgenic parental plant line increased with a composition comprising the selective agent to which the resistance gene confers resistance.
2. 2. The method according to claim 1, characterized in that the sterility gene is selected from the group consisting of p.AN::Tox, dam methylase, or Barnase.
3. 3. The method of compliance with the claim 1, characterized in that the resistance gene confers resistance to a herbicide.
4. The method according to claim 3, characterized in that the herbicide resistance gene confers resistance to a selective agent, comprising a herbicide selected from the group consisting of inhibitors of amino acid synthesis, photosynthesis inhibitors, lipid inhibitors, growth regulators, cell membrane switches, pigment inhibitors and growth sowing inhibitors.
5. 5. The method according to claim 4, characterized in that the herbicide resistance gene confers resistance to a selective agent, comprising a herbicide selected from the group consisting of imidazolinones, sulfonylureas, triazolopyrimidines, glyphosate, sethoxydim, fenoxaprop, glufosinate, the triazines and bromoxynil.
6. The method according to claim 3, characterized in that the herbicide resistance gene is selected from the group consisting of Bar, PAT, aroA, Epsps, Corl-1, bxn, and psbA.
7. 7. The method according to claim 1, characterized in that the resistance gene confers resistance to an antibiotic.
8. 8. The method of compliance with the claim 7, characterized in that the antibiotic resistance gene confers resistance to a selective agent comprising an aminoglycoside antibiotic.
9. The method according to claim 8, characterized in that the antibiotic resistance gene confers resistance to a selective agent comprising an aminoglycoside antibiotic selected from the group consisting of kanamycin, gentamicin, G418, neomycin, paromycin, and hygromycin.
10. 10. The method in accordance with the claim 7, characterized in that the antibiotic resistance gene is selected from the group consisting of the NPT II gene, the aphA2 gene, and the hpt-aphlV gene.
11. 11. The method according to the claim I, characterized in that the resistance gene confers resistance to a selective agent comprising a chemical, toxic other than a herbicide or an antibiotic.
12. 12. The method according to claim 11, characterized in that the toxic chemical comprises a toxic metabolite.
13. 13. The method according to claim 12, characterized in that the toxic metabolite is 4-methyltryptophan.
14. 14. The method according to the claim II, characterized in that the resistance gene is the tryptophan decarboxylase gene.
15. The method according to claim 1, characterized in that the transgenic parental plant line further comprises a constitutive promoter linked to the resistance gene.
16. 16. The method according to claim 15, characterized in that the constitutive promoter is selected from the group consisting of CaMV 19S, CaMV 35S, CaMV double 35S, ALS, MAS, and ubiquitin.
17. 17. The method in accordance with the claim 1, characterized in that the transgenic paternal-plant line also comprises a chemically inducible gene that controls the expression of the resistance gene.
18. 18. The method of compliance with the claim 17, characterized in that the chemically inducible gene is induced by a hormone.
19. 19. The method according to the claim 18, characterized in that the hormone is a steroidal hormone.
20. 20. The method according to claim 17, characterized in that the chemically inducible gene is induced through a chemical without hormone.
21. 21. The method according to claim 20, characterized in that the chemical without hormone is an insurer.
22. 22. The method according to claim 1, characterized in that the transgenic parental plant line further comprises at least one tissue-specific promoter.
23. 23. The method according to claim 22, characterized in that the tissue-specific promoter is a seed-specific promoter.
24. The method according to claim 17, characterized in that the seed of the increased transgenic parental plant line is coated with a composition comprising an effective amount of the chemical that induces the chemically inducible gene.
25. 25. The method according to claim 24, characterized in that the chemical agent that induces the chemically inducible gene is selected from the group consisting of hormones and insurers.
26. 26. The method according to claim 1 or claim 24, further characterized by comprising planting the coated seed, growing mature plants, fertilizing mature plants, and harvesting the seeds of the mature plants.
27. 27. Mature plants produced by the method according to claim 26.
28. 28. Seeds of plants produced through the mature plants, according to claim 27.
29. 29. A method to maintain male sterility in plants, the method is characterized in that it comprises generating a transgenic paternal plant line, the transgenic paternal plant line comprises a male sterility gene, genetically linked to a resistance gene, which confers resistance to a selective agent, the resistance gene is under the control of a chemically inducible gene; increase the line of transgenic paternal plant; and coating the seed of the increased transgenic parental plant line with a composition comprising - effective amounts of the selective agent to which the resistance gene confers resistance and the chemical agent to which the chemically inducible gene responds.
30. 30. A method to maintain male sterility in plants, the method is characterized in that it comprises generating a transgenic paternal plant line, the transgenic parental plant line comprises a male sterility gene, genetically linked to a resistance gene, which confers resistance to a selective agent, the resistance gene is under the control of a seed-specific promoter; increase the line of transgenic paternal plant; and coating the seed of the increased transgenic parental plant line with a composition comprising an effective amount of the selective agent to which the resistance agent confers resistance.