MXPA05010675A - Modulation of cytokinin activity in plants - Google Patents

Abstract

This invention relates generally to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for the temporally- and/or spatially-regulated expression of genes that affect metabolically effective levels of cytokinins in plants, particularly in plant seeds and related female reproductive tissue. This invention further relates to transgenic plants having enhanced levels of cytokinin expression wherein the transgenic plant exhibits useful characteristics, such as improved seed size, decreased tip kernel abortion, increased seed set during unfavorable environmental conditions, or stability of yield. The present invention also provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions comprise novel nucleotide sequences for seed-preferred promoters known as eep1 and eep2. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one of the promoters of the present invention and regenerating a stably transformed plant from the transformed plant cell.

Description

MODULATION OF THE ACTIVITY OF CYTOKININES IN PLANTS Field of the invention: This invention relates in general to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for the regulated expression in time and space of genes that affect the metabolically effective levels of cytokinins in plants, including seeds and the maternal tissue from which said seeds come, including inflorescences. female, ovarian, female, female, aleurone, pedicel and pedicel-forming regions.

BACKGROUND OF THE INVENTION: Cytokinins are phytohormones that are involved in numerous physiological processes in plants. Plants respond to different types of environmental stress in part by modifying the relative balance of active and inactive cytokinins. For example, during some type of abiotic stress (including, for example, conditions of drought, density, cold, salinity and / or soil compaction), increased cytokinin oxidase activity displaces the balance in favor of inactive cytokinins, which leads to lower plant productivity. (Jones and Setter, in CSSA Special Publication No. 29, pp. 25-42, American Society of Agronomy, Madison, Wl. (1999)). Conversely, a directed manipulation of the cytokinin balance in favor of active cytokinins could result in higher productivity, even under conditions of abiotic stress, through mechanisms such as increased cell division, induction of stomatal opening, inhibition of senescence of organs and / or suppression of apical dominance. (Morris, R.O., 1997, in Cellular and Molecular Biology of Plant Seed Development, pp. 117-148, Kluwer Academic Publishers. (1997)). It has been shown that cytokinins decrease in maize subjected to unfavorable environmental conditions, resulting in a reduced seed size, increased number of abortions of young grains and less establishment of seeds. (Cheikh and Jones, Plant Physiol., 106: 45-51 (1994); Dietrich et al., Plant Physiol Biochem 33: 327-336 (1995)). Therefore, these studies show that under stress conditions an approach to improve the establishment of seeds and the size of the seeds could be the maintenance of the cytokinin group. active above a critical threshold level. The first natural cytokinin was purified in 1963 (Letham, DS, Life Sci. 8: 569-573 (1963)) from immature grains of Zea mays and identified as 6- (4-hydroxy-3-methylbut-trans- 2-enylamino) purine, better known today as zeatin. On average, all natural cytokinins appear to be purine derivatives with a branched substituent N6 carbon 5. (See: McGaw, BA, In: Plant Hormones and their Role in Plant Growth and Development, ed. PJ Davies, Martinus Nijhoff Publ., Boston, 1987, Chapter B3, pp. 76-93, whose content is incorporated in this document as background for reference). Although some 25 different natural cytokinins have been identified, those that are considered particularly active are N6 (? 2-isopentenyl) adenosine (iP), zeatin (Z), diHZ, benzyladenine (BAP) and the 9-ribosyl derivatives (and, in the case of Z and diHZ, O-glucosyl) thereof. However, said activity is markedly reduced in conjugates 7- and 9-glucosyl and 9-alanyl. These latter compounds may be the reflection of deactivation or control mechanisms. The metabolism of cytokinins in plants is complex. Multi-step biochemical pathways for the biosynthesis and degradation of cytokinins are known. At least two major routes of cytokinin biosynthesis are recognized. The first comprises a transfer RNA (tRNA) as an intermediate. The second comprises enzymes that catalyze the formation of cytokinins through de novo (direct) biosynthesis. In the first case, it is known that tRNAs contain various hypermodified bases (including certain cytokinins). It is also known that these modifications appear at the level of the tRNA polymer as a post-modification. transcription. The branched substituent N6 carbon 5 is derived from mevalonic acid pyrophosphate, which undergoes decarboxylation, dehydration and isomerization processes to result in? 2-isopentenyl pyrophosphate (iPP). The latter is condensed with the relevant adenosine residue in the tRNA. Then other modifications are possible. Finally, the tRNAs are hydrolyzed in the bases that compose them, forming in this way the grouping of available free cytokinins. Alternatively, enzymes have been discovered that catalyze the formation of de novo cytokinins, that is, without an intermediate tRNA. The ipt gene used in the practice of this invention is one such gene. It is considered that the formation of free cytokinins starts with [9R5'P] iP. This compound is hydroxylated very rapidly and stereospecifically to result in the zeatin derivatives from which numerous additional metabolic events may arise. Such events include, for example (1) conjugation, with incorporation of ribosides, ribotides, glycosides and amino acids; (2) hydrolysis; (3) reduction; and (4) oxidation. In particular, it is known that O-glycosylation of zeatin is important in the regulation of the level of active cytokinins. Although each of the enzymes of these pathways may be a candidate as an effector of cytokinin levels, the enzymes associated with the rate-limiting steps are of particular utility in the practice of this invention. One such enzyme is isopentenyl transferase (ipt). The isolated gene encoding ipt was described by van Larebeke et al., Nature 252: 169-170 (1974)); see also Barry et al., Proc. Nat'l. Acad. Sci. (USA) 81: 4776-4780 (1984) and Strabala et al., Mol. Gen. Gen. 216 (2-3): 388-394 (1989).

The isolation of ipt genes in Arabidopsis has also been reported. (Takei et al., J Biol Chem. 276 (28): 26405-26410 (2001), Kakimoto et al., Plant Cell Physiol. 42 (7): 677-685 (2001) and WO 2002/072818; Sun et al. al., Plant Physiol 131: 167-176 (2003)). The invention comprises the expression of appropriately modulated ipt genes from any source, including other species, such as corn. Based on the demonstrable effects of cytokinins in hundreds of experiments on multiple plant species, it was considered that a transgenic approach to increase active cytokinins in maize could improve productivity under normal conditions and / or abiotic stress. However, a simple increase in the group of active cytokinins does not automatically lead to a better growth of the plants. In fact, it has been shown that the elevation of cytokinin levels generates harmful effects on the phenotype of plants. For example, Smigocki et al. . { Proc. Nati Acad. Sci. (USA) 85: 5131-5135 (1988)), demonstrated, using the ipt gene of A. tumefaciens operably linked to the 35S or NOS promoter, a generalized effect on shoot organogenesis and zeatin levels . It was observed that the activity of the promoter controls the degree of morphogenic response observed and an unregulated production of cytokinins can result in undesired pleiotropic effects. With the constructions just mentioned, unwanted effects included a complete inhibition of root formation in tobacco, and squat cucumber seedlings that did not survive. (Smigocki et al., Klee et al., Annual Rev. Plant Physiol., 38: 467-486 (1987)).

Other attempts followed to express the ipt gene in a more controlled manner. Medford et al. . { The Plant Cell 1: 403-413 (1989)) described the Agrobacterium ipt gene under the control of a heat inducible hsp70 promoter and the expression thereof in transgenic tobacco plants. Cytokinin levels increased dramatically after heat treatment and the effects observed in transgenics included significant reductions in height, xylem content and leaf size. Both in tobacco and in Arabidopsis, the transgenic ones showed a slower root growth, a disordered development of roots and a greater growth of axillary buds in relation to the wild type plants. In addition, the experimental constructions were not satisfactory because the plants presented phenotypes associated with an excess in cytokinin levels, including a lower height, leaf area and stem width, even in the absence of thermal induction. In addition, certain changes were observed in both wild-type and transgenic plants and could be attributed to heat induction per se. Schmulling, T. et al., (FEBS Letters, 249 (2): 401-406 (1989)) transformed tobacco with the Agrobacterium ipt gene under the control of the Drosophila hsp70 promoter, which provides a very low level of expression to normal temperatures and a rapid increase in expression after heat shock. Most transgenic callus that suffered heat shock were greener, had higher concentrations of cytokinins and grew at a faster rate than control calluses. Plants regenerated from transgenic calluses subjected to heat shock were described as "fairly normal" and the levels of cytokinins in these plants did not differ from those that were measured in wild-type plants. The plants regenerated from non-induced transgenic callus did not differ from the controls in terms of plant phenotype or cytokinin content. A second experiment allowed to create transgenic callus tissue for the ipt gene directed by its native promoter. In regenerated shoots from these calluses, high levels of cytokinins inhibited root formation. These shoots, grafted onto stems of wild-type tobacco, presented tiny leaves and a habit of stunted, highly branched growth. Therefore, the transformation resulted in negative phenotypic changes or had no impact. In PCT Patent Application Publication No. WO 91/01323, February 7, 1991, and US Pat. No. 5,177,307 and 4,943,674, tomato plants transformed with the ipt gene linked to specific promoters of fruits (2AII, Z130 and Z70) that had modified ripening characteristics are described. The fruits were described as rough in immature stages and as mottled, spotted and uneven during ripening. See also U.S. Pat. No. 6,329,570, which describes the transformation of cotton with ipt and a promoter with preference for seed tissues to modify the establishment of capsules and the quality of the fibers. In PCT Patent Application Publication No. WO 93/07272, the ipt gene was fused with the chalcone synthetase (chs) promoter of Antirrhinum majus and expressed in potato. The phenotypic alterations of the transformants included a higher yield of tubers, height of the plants and size of the leaves *, thicker stems and delayed leaf senescence. Wang et al. (Australlan J of Plant Phys 24 (5): 661-672 and 673-683, 1997) reported Increased levels of cytokinins in leaf blades and upper stems of tobacco transformed with ipt directed by a chs promoter, as well as the release of axillary buds, inhibition of root development, retardation of leaf senescence, elevation of chlorophyll levels, delay in the beginning of flowering, retardation of flower development, growth of leaf shoots from the primary root, change in the shape of the leaves, central veins of the larger leaves, enlarged veins, thicker stems, greater number of nodes and a higher rate of perspiration. The expression of chalcone synthetase genes is complex and is regulated by numerous factors, including light, generators of fungal responses, lesions and microbial pathogens. In addition, the expression of chs may have preference for tissues, taking place mostly in roots and pigmented flowers, and be developmental specific, taking place during early germination. (Ito et al., Mol. Gen. Gen. 255: 28-37 (9997); Shimizu et al., Plant Molecular Biology 39 (4) 785-95 (1999)). Other gen / pir / promoter constructs have been described. Smigocki et al., In WO 94124848 and U.S. Pat. No. 5,496,732 and 5,792,934, described a genetic construct capable of conferring increased insect resistance comprising an injury-inducible promoter fused to an ipt gene. The study focused on insect resistance and no changes in plant morphology are described. Houck et al., In U.S. Pat. No. 4,943,674 and 5,177,307, described several promoters (2AII, Z130 and Z70) coupled to genes encoding enzymes of the metabolic pathway of cytokinins, in particular ipt, for the expression of said enzymes in tomato fruits.

Amasino et al., In PCT Patent Application Publication WO 96/29858 disclose two specific promoters of senescence, including SAG12, operably linked to an ipt gene to inhibit foliar senescence in tobacco. The transformants developed normally, with higher biomass and flower and seed production, perhaps due to the extended development period created by the delay in senescence. See also: US Patents N °: 5,689,042 and 6,359,197; Gan, S. et al., Science 270: 1986-1988 (1995). Jordi et al., Plant, Cell and Environment 23 (3): 279-289 (2000), studied the physiological effects of SAG12: / pí construction in tobacco. Although the older leaves benefited by retaining chlorophyll, Rubisco and proteins, the remobilization of nutrients from the older leaves to the younger leaves was possibly reduced, which led to limited photosynthesis in the upper leaves and restrictions in the potential increase of leaves. the biomass of these plants, particularly under stress conditions. Roeckel, P. et al., (Transgenic Res. 6 (2): 133-141 (1997)) transformed canola and tobacco with an ipt gene under the control of the 2S albumin promoter regulated by the development, specific to Agrobacterium seeds . Although only ipt mRNA was found in seeds, and cytokinin levels were only evaluated in seeds, the effects of construction were not limited to seeds: tobacco plants had reduced roots; the cañola plants were "surprisingly" (p.139) taller and more branched and had more seed bearing structures. However, the yield was not affected, nor the type of leaf, number of leaves, days until the first flower or days until sprouting, in both species.

The transformation of tobacco with ipt bound to a promoter inducible by copper, specific of roots, provided, in 28 of 31 cases, a controlled system to evaluate the effects of a greater production of cytokinins. Morphological changes with induction included release of apical dominance, increases in the total number of leaves of the plants and delay of leaf senescence. (McKenzie et al., Plant Physiol. 116: 969-977 (1998)) However, numerous transgenic lines exhibited an uncontrolled expression of cytokinins and a radically different, unwanted phenotype that lacked root development and stem elongation. Ivic et al. (Plant Cell Reports 20: 770-773 (2001)) reported that expression of ipt in transgenic sugar beet resulted in severe inhibition in root development, together with undesired changes in leaf and shoot morphology. The transformed seedlings formed roots very slowly or did not form them and had a very low survival rate when transferred to land. Sa et al. (Transgenic Research 11 (3): 269-278, 2002) reported that the transformation of tobacco with Agrobacterium ipt under the control of a TA29 promoter, which is specifically expressed in anthers, resulted in a disturbance in the development of anthers and pollen. Approximately 80% of the T0 transgenic plants exhibited a significant decrease in the pollen germination index and up to 20% of the T0 transgenic plants showed male sterility. In addition, abnormal pistils and stamens were found in the transgenic plants. Such negative effects due to a targeted expression of transgenic IPT were described in PCT Publication WO 00/52169: "These approaches also produce unwanted side effects in the plant and, even in cases where the ipt or rolC genes are expressed under the control of tissue-specific promoters, these side effects are observed in other tissues, probably because the cytokinins are easily transported between cells and tissues of the plant. "(the commendation is ours) Therefore, there still persists the need for nucleic acid constructions and methods that are useful to control and direct a regulated expression in time and space of the cells. metabolic cytokinin genes in plants, including the seeds of plants and those maternal tissues in which seed development takes place, or to modulate the sensitivity and / or response of plants to cytokinins, in order to improve the vigor and yield of plants without harmful effects such as reduced root development or aberrant outbreak morphology. invention provides several such nucleic acid constructs and methods for modulating the activity of cytokinins in plants, including effective levels of cytokinins in the seeds of plants, in developing seeds, and in related reproductive tissues. In addition, the need persists for constructions and methods that provide such improvements in the vigor and yield of the plants under favorable or unfavorable growth conditions. This invention provides tools and reagents that allow the specialist, with the application, among others, of transgenic methodologies, to affect the level of activity of cytokinins, including the metabolic flux with respect to the metabolic pathway of cytokinins in seeds. This effect can be anabolic or catabolic, with which reference is made to the fact that the effect can act by increasing the biosynthesis of cytokinins and / or decrease degradation. This invention also contemplates a combination of both approaches. Other combinations may include a directed modulation of the expression of isolated polynucleotides encoding the polypeptides involved in the recognition of cytokinins and the cellular response to provide increased activity of the cytokinins defined herein.

SUMMARY OF THE INVENTION Certain embodiments of the present invention provide plants, in particular transgenic maize, which exhibit increased cytokinin activity, relative to an otherwise isogenic plant, without the corresponding deleterious effects. Said increase in relation to said otherwise isogenic plant can take place under favorable environmental conditions, unfavorable environmental conditions or both. The increased activity of cytokinins can encompass levels of cytokinins in seeds, in developing seeds and in maternal tissues associated with the development of seeds. As an alternative, or in addition, the increased cytokinin activity may be the result of a greater perception of, and response to, the cytokinins by said plant. The increased cytokinin activity can act as a metabolic buffer to alleviate the effects of different types of transient stress, particularly during the retardation phase of seed development, to consequently improve stress tolerance and corn yield stability. The increased cytokinin activity can also be manifested by a greater vigor of the plants and / or a higher yield of seeds. Said embodiments comprise a nucleic acid construct stably integrated into the genome of the plant, wherein said construct can temporally or spatially modulate cytokinin levels. Certain embodiments of the present invention provide lines of transgenic plants with inheritable phenotypes that are useful in breeding programs designed to produce commercial products with a higher yield, which may include plant vigor, larger seed size, lower amount of young grain abortion and / or greater establishment of seeds under favorable or unfavorable environmental conditions. Said commercial products constitute additional embodiments of the invention. Some embodiments of the invention provide a fertile transgenic plant comprising a nucleic acid construct stably integrated into the genome thereof, wherein said construction allows to modulate the activity of the cytokinins in said plants. Certain embodiments of the invention provide a molecule of Isolated recombinant DNA comprising a promoter that directs a temporally or spatially regulated expression operably linked to a cytokinin modulator gene and optionally comprising one or more enhancer elements of a gene of high expression. In some embodiments, the invention provides a method for improving stress tolerance and yield stability in plants, comprising stably introducing into a plant cell a nucleic acid construct capable of modulating cytokinin activity and, from said cells, regenerate said plants with greater stress tolerance and yield stability. Said construction can result in a preferential expression of the cytokinin modulator genes during the delay phase of seed development. The embodiments also provide a method for producing fertile transgenic plants capable of providing regulated expression of a cytokinin modulator gene in developing seeds, comprising introducing in plant host cells a nucleic acid construct that allows a preferential temporal and / or spatial expression of a cytokinin modulator gene in developing seeds and in the maternal tissues associated with the development of the seeds, under conditions sufficient for a stable integration of the construction in the genome of said cells and regenerate and recover said transgenic fertile plants. Other embodiments of the invention provide a method for producing fertile transgenic plants with greater vigor, which comprises introducing a nucleic acid construct into plant host cells that allows the modulation of cytokinin activity., under sufficient conditions for a stable integration of said construction in the genome of said cells and regenerate and recover said fertile transgenic plants. In accordance with these aspects of the invention, isolated nucleic acid molecules encoding cytokinin metabolism enzymes are provided, including mRNA, cDNA, genomic DNA and variants, analogs or derivatives of biological utility thereof, including fragments of said variants, analogues and derivatives. Other embodiments of the invention are natural allelic variants of the nucleic acid molecules in the provided sequences encoding enzymes of cytokinin metabolism. Also provided are polypeptides comprising cytokinin metabolism enzymes as well as fragments of biological utility or diagnostics thereof, as well as variants, derivatives and analogues thereof and fragments thereof. For example, cytokinin metabolism polypeptides, in particular ipt (e.g., SEQ ID N °: 1 and 2) and cytokinin oxidase (e.g., SEQ ID N °: 26-37), are specifically provided. employ for modulating cytokinin levels in related female reproductive tissues and seeds, particularly the meristematic regions of female reproductive tissues. Certain embodiments of the invention provide methods for producing the polypeptides of interest, which comprises culturing host cells that have incorporated for their expression a polynucleotide under conditions that allow a temporal and / or spatial expression of the enzymes of cytokinin metabolism in seeds and reproductive tissues. related females, and then optionally recover the expressed polypeptide. Furthermore, in certain embodiments, probes are provided that hybridize with the cytokinin metabolism enzyme polynucleotide sequences useful as molecular markers in breeding programs. Other embodiments of the invention provide products, compositions, processes and methods that use the aforementioned polypeptides and polynucleotides for research, biological and agronomic purposes. Other embodiments of the invention provide inhibitors of said polypeptides, useful for modulating the activity and / or expression of the polypeptides. In particular, antibodies against said polypeptides are provided. In certain embodiments of this aspect of the invention, antibodies against cytokinin catabolic enzymes are provided. The antibodies can be selective for the entire class of cytokinin catabolic enzymes, independently of the species of origin, as well as species-specific antibodies. Still other embodiments provide antagonists and agonists of cytokinin enzymes. Among the preferred antagonists, those which are they bind cytokinin catabolic enzymes (eg, to cytokinin oxidase) in order to inhibit the binding of the binding molecules, or to destabilize the complex formed between the cytokinin catabolic enzyme and the binding molecule, in order to to prevent any biological activity that could be due to said cytokinin catabolic enzyme. Preferred agonists include molecules that bind to or interact with cytokinin biosynthesis enzymes in order to stimulate one or more effects of a particular cytokinin biosynthesis enzyme or that enhances the expression of the enzyme and that also preferably results in the modulation of cytokinin accumulation. Effective constructions result in the modulation of cytokinins in the meristematic tissues, in particular in the female reproductive tissues, thus providing the observed improvement in vigor. The invention encompasses the particular constructions described herein and other similar constructs that can provide expression of cytokinin-modulating genes to result in increased vigor of the plants without significant deleterious effects. In any case, and without being limited to any particular theory, the modulation of the activity of cytokinins in the female reproductive tissues of said plant is claimed, to obtain as a result a greater vigor of the plants without significant deleterious effects. Expression of isolated DNA sequences in a plant host depends on the presence of operatively linked regulatory elements that are functional within the plant host. The choice of regulatory sequences will determine when and where within the organism that is will express the isolated DNA sequence. When continuous expression is desired in all or almost all the cells of a plant during development, constitutive promoters will be used. Conversely, when gene expression is desired in response to a stimulus, inducible promoters are the regulatory element of choice. When expression in particular tissues or organs is desired, sometimes at specific stages of development, promoters and / or terminators are preferably used for tissues. That is, these regulatory elements can direct expression in specific tissues or organs, in specific stages. Other 5 'and / or 3' regulatory sequences can be included with respect to the central sequences in the expression cassettes of the transformation vectors to obtain variable levels of expression of the isolated nucleotide sequences in a transgenic plant.

Seed development comprises embryogenesis and maturation events, as well as physiological adaptation processes that take place within the seeds to ensure the survival of the progeny. Developing seeds accumulate and store carbohydrates, lipids and proteins that will later be used during germination. In general, the expression patterns of the seed proteins are highly regulated. This regulation includes spatial and temporal regulation during seed development. Numerous proteins accumulate and decay during embryogenesis and seed development and provide an excellent system to investigate different aspects of genetic regulation, as well as to provide regulatory sequences that can be used in the genetic manipulation of plants. As the field of plant bioengineering develops, and that there is access to a greater number of genes, there is an increasing need to transform multiple genes. These multiple exogenous genes typically need control by separate regulatory sequences. Some genes must be regulated constitutively, while other genes must be expressed at certain stages of development or locations in the transgenic organism. Therefore, various regulatory sequences with different effects will be needed. Another reason why various regulatory sequences are needed is that unwanted biochemical interactions may occur with the use of the same regulatory sequence to control more than one gene. For example, the transformation with multiple copies of a regulatory element can cause a homologous recombination between two or more expression systems, the formation of fork loops from two copies of the same promoter or enhancer very close in opposite orientation, competition between systems of identical expression by binding to common promoter-specific regulatory factors and inappropriate expression levels of an exogenous gene due to trans effects of a second promoter or enhancer. In view of these considerations, one of the goals in this field has been the detection and characterization of new regulatory sequences for the transgenic control of DNA constructions. The isolation and characterization of promoters and terminators with preference for seeds that can serve as regulatory elements for the expression of isolated sequences of the nucleotides of interest in a preferred manner for seeds are necessary to improve the characteristics of the seeds in plants. In particular, early grain development constitutes a critical stage in abortions of young grains induced by drought. It has been proved that the maintenance of an active group of plant cytokinins is critical to sustain the growth and development of the grains under transient conditions of drought stress. In addition, the genes that contribute to general stress responses, such as those that are involved in ABA responses, and also the genes that maintain cell expansion and division, fulfill fundamental functions in reproductive development under stress. The endosperm of the early stages have emerged as an important target tissue for transgenic expression since it surrounds and nourishes developing embryos. Promoters EEP1 and EEP2 refer to the need to direct transgenic expression in early endospermic tissue. Other objects, features, advantages and aspects of the present invention will be apparent to the specialists from the following description. It should be kept in mind, however, that the following description and specific examples, although indicative of the preferred embodiments of the invention, are offered only by way of illustration. The various changes and modifications that are within the spirit and scope of the invention described will be evident to those skilled in the art from reading the following description and from reading the other parts of the present.

Brief description of the figures: Figure 1 A-Embryo: This figure shows that a preferred overexpression in ipt embryos increases the levels of cytokinins in the embryo, in particular of ZR and Z9G (a difference in the range of 2 to 8 times). On the contrary, Z levels remain unchanged and I PAR is not detectable at any stage of development. Abbreviations: Z = zeatin, ZR (or [9R] Z) = zeatin riboside, Z9G (or [9GJZ) = zeatin-9-glucoside, IPA or [9R] iP = isopentenyldenosine, IPAR (or [9R-5'P] iP) = isopentenyladenosine-5'-monophosphate and DAP = Days after pollination. Figure 1 B-Endosperm: This figure shows that an overexpression of ipt preferred in the embryo altered the levels of cytokinins in the endosperm but less than the levels in the embryo (a difference in the range of only 10 to 30% ). The abbreviations of Figure 1A were used. Figure 2 shows the data of the growth index of ears for hemicigote plants D2F1 under non-stressing conditions. Figure 3 shows yield data of the grains, the amount of grains, the dry mass of the grains and the length of the cobs for hemicigote plants D3F1 under non-stressing conditions. Figure 4 shows plant height data for homozygous D4F3 plants under non-stressing conditions. Figure 5 shows the yield data for homozygous D4F3 plants under non-stressing conditions. In Figure 6 the data of the yield components for homozygous D4F3 plants are provided under non-stressing conditions.

In Figure 7, plant height data for D4F3 plants subjected to drought stress are provided. Figure 8 provides green leaf data for D4F3 plants subjected to drought stress. Figure 9 provides yield data for D4F3 plants subjected to drought stress. Figure 10 shows the increase in plant biomass for event TC15850.

Glossary: The following illustrative explanations are provided to facilitate the understanding of certain terms frequently used in the present, particularly in the examples. Said explanations are provided for greater convenience and not for limiting the invention. ACTIVITY OF CYTOKININES, as used herein, encompasses active cytokinin levels within a plant, as well as the sensitivity and response of plants to cytokinins. Therefore, enzymes of cytokinin biosynthesis and cytokinin degrading enzymes are examples of enzymes capable of modulating cytokinin activity. A "cytokinin modulator gene" comprises polynucleotides that encode such enzymes, as well as polynucleotides that encode proteins compromised in cytokinin sensitivity and the response of plants, including transcription factors associated with the response to cytokinin. A "pool of active cytokinins" refers to the accumulation of active cytokinins at any time within a cell or part of the whole plant or plant, as appropriate. The stabilization of the group of active cytokinins can comprise the subsensitization of the degradation or conjugation of cytokinins or the sensitization of cytokinin biosynthesis. ENZYMES UNION MOLECULE OF METABOLISM OF CYTOKININS, as used herein, refer to molecules or ions that specifically bind or interact with the polypeptides or polynucleotides of the cytokinin metabolism enzymes of the present invention, including, for example, enzyme substrates, membrane components cellular and classic receivers. The binding between the polypeptides of the invention and said molecules, including binding or interaction molecules, may be unique to the polypeptides of the invention or may be highly specific for the polypeptides of the invention or may be highly specific for a group of proteins that includes the polypeptides of the invention or it may be specific for several groups of proteins of which at least it includes a polypeptide of the invention. Binding molecules also include antibodies and reagents derived from antibodies that specifically bind to the polypeptides of the invention. COMPONENT RESPONDING TO CYTOKININES, as used herein, generally means a cellular constituent that binds or otherwise interacts with a cytokinin resulting in the transmission of an intra or intercellular signal and the generation of one or more cellular responses to the presence or absence or fluctuation of cytokinin levels. PLANT DEVELOPMENT SEEDS, as used herein, refer in general to maternal plant tissues that after pollination have the capacity to generate a vegetable seed. This maternal plant tissue includes tissues such as female florets, ovaries, aleurone, pedicels and pedicel-forming regions. The HARMFUL effects, as considered in general and as used herein, are those that are obviously harmful or harmful. The significant adverse effects, in the context of this application, refer to the phenotypic changes that could contribute with a net negative effect for the productivity or vigor of the plants. SILENCING GENES, refers to a post-interference transcription with the genetic expression. Techniques such as, for example, antisense, cosuppression and RNA interference (RNAi) have been shown to be effective for gene silencing. (For reviews on the subject, see Amdt and Rank, Genome 40 (6): 785-797, 1997; Turner and Schuch, Journal of Chemical Technology and Biotechnology 75 (10): 869-882, 2000; Klink and Wolniak, Journal of Plant Growth Regulation 19 (4): 371-384, 2000) GENETIC ELEMENT, as used herein, generally refers to a polynucleotide comprising a coding region of a polypeptide, or a region of a polynucleotide that regulates replication, transcription or translation or other processes important for the expression of the polypeptide in a host cell, or a polynucleotide comprising both a coding region of a polypeptide and a region operatively linked thereto that regulates expression. The genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the genome of the host cell. They may be comprised within plasmids. Genetic elements may also be comprised within the genome of a host cell; not in its natural state but, rather, after a manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others. GERMPLASM, as used herein, refers to a set of genetic entities, which can be used in a breeding program to develop new plant varieties. HIGH CONTENT OF TRANSGENIC CYTOKININS, as used herein, refers to an entity, which, as a result of a Recombinant genetic manipulation, produces seeds with an increase in cytokinins and / or a decrease in hereditary auxins. HOST CELL, as used herein, is a cell that has been transformed or transfected or that can be transformed or transfected by an exogenous polynucleotide sequence. An "exogenous polynucleotide sequence" is defined as a sequence that is not naturally found in the cell or that is naturally present in the cell but at a different genetic locus, with a different copy quantity or under the direction of a different regulatory element . IDENTITY AND SIMILARITY, as used herein, and as known in the art, are relationships between two polypeptide sequences or two polynucleotide sequences, determined by comparison of sequences. In the art, identity also refers to the degree of sequence relationship between two polypeptides or two polynucleotide sequences determined by the coincidence between two chains of said sequences. Both identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York , 1993, Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994, Seqúense Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Methods commonly employed to determine the identity or similarity between two sequences include, for example, those described in Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods Preferred to determine identity are designed to obtain the greatest agreement between the two evaluated sequences. The methods to determine identity and similarity are encoded in computer programs. Typical computer program methods for determining identity and similarity between two sequences include: the GCG® program package (Accelrys, Inc., San Diego, CA; Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA and TFASTA (Atschul, SF et al., J. Mol. Biol. 215: 403 (1990) ). ISOLATED, as used herein, means altered "by the hand of man" with respect to its natural state; that is, if it appears in nature, it has been changed or separated from its original environment or both. For example, a polynucleotide or natural polypeptide usually present in a living organism in its natural state is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is used in the present. For example, with respect to polynucleotides, the term "isolated" means that it is separated from the chromosome and from the cell in which it occurs naturally. As part of or after isolation, said polynucleotides can be linked to other polynucleotides, such as, for example, DNA, for mutagenesis, to form fusion proteins and for propagation or expression in a host. Isolated polynucleotides, either alone or linked to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Once introduced into host cells in culture or in whole organisms, said DNA would still be isolated, as that term is used herein, because they would not be found in their natural form or environment.

Similarly, polynucleotides and polypeptides may appear in a composition, such as formulations or solutions of media for introduction into cells, or compositions or solutions for chemical or enzymatic reactions, which are not natural compositions and in them polynucleotides or polypeptides they remain isolated according to the meaning of said term as used herein. LIGATION, as used herein, refers to the process of forming phosphodiester bonds between two or more polynucleotides, which very often are double-stranded DNA. The ligation techniques are well known in the art and the ligation protocols are described in the laboratory manuals and standard references, such as, for example, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al., P. 146, cited below. LOW LEVEL OF CONSTITUTIVE EXPRESSION refers to the. genetic expression in essentially all tissues of a plant and in most or all stages of development, at a level lower than that of a gene driven by the CaMV3dS promoter. The low level of constitutive expression of a polynucleotide can be the result of operative ligation to a promoter that normally directs such expression, such as F3.7 (SEQ ID NO: 10) or of a combination of a promoter operably linked to a gene, wherein said combination is also close to an enhancer element, such as the CaMV3ds enhancer. (See, for example, Mol. Gen. Gen. 261: 635-643 (1999)). Promoters that direct expression preferentially in meristematic tissues, such as zag2.1 (SEQ ID NO: 3), may also provide a low level of constitutive expression.

OLIGONUCLEOTIDE (S), as used herein, refers to short polynucleotides. This term often refers to single chain deoxyribonucleotides, but may also refer to single or double stranded ribonucleotides, RNA: DNA and double stranded DNA hybrids, among others. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized using chemical methods, such as those implemented in automated oligonucleotide synthesizers. However, oligonucleotides can be obtained using numerous additional methods, including techniques mediated by recombinant DNA in vitro and expression of DNA in cells and organisms. Initially, chemically synthesized DNAs are typically obtained without a 5 'phosphate. The 5 'ends of said oligonucleotides are not substrates for phosphodiester linkage formation by ligation reactions employing the DNA ligases typically used to form recombinant DNA molecules. When it is desired to bind said oligonucleotides, a phosphate may be added by standard techniques, such as those employing a kinase and ATP. The 3 'end of a chemically synthesized oligonucleotide generally has a hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, a phosphodiester linkage will be easily formed with the 5' phosphate of another polynucleotide, such as another oligonucleotide. As is generally known, it is possible to prevent this reaction selectively, when desired, by separating the 5 'phosphates from the polynucleotide (s) before ligation. OPERATIONAL LINKAGE, as used herein, includes reference to a functional linkage between a promoter and a second sequence, wherein said promoter sequence initiates and intervenes in the transcription of the DNA corresponding to the second sequence. In general, "operably linked" means that the ligated nucleic acid sequences are contiguous and, when it is necessary to join two coding regions of contiguous proteins, in the same reading frame. PLANT, as used herein, includes references to complete plants, parts or organs of plants (for example, leaves, stems, roots, etc.), plant cells, seeds and progeny thereof. A plant cell, as used herein, also includes, without limitation, the cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes , pollen and microspores. Plant cells can also include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants that is amenable to transformation techniques, including monocotyledonous and dicotyledonous plants, including, for example, corn, soybeans and canola . PLASMIDS, as used herein, are herein generally designated with a lowercase letter "p" preceded and / or followed by capital letters and / or numbers, in accordance with standard naming conventions that are familiar to those skilled in the art. . The initial plasmids described herein are commercially available, available to the public, or can be constructed from the available plasmids by routinely applying published and recognized procedures. Many of the plasmids and other cloning and expression vectors that can be used according to the present invention are well known and readily available to those skilled in the art. Moreover, specialists can construct any of the numerous plasmids suitable for use in the invention. The properties, construction and use of said plasmids, as well as other vectors, in the present invention will be apparent to the specialists from the present description. POLYUCLEOTIDE (S), as used herein, refers in general to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, the term polynucleotides, as used herein, refers, inter alia, to single and double stranded DNA, DNA which is a mixture of single and double stranded regions or single stranded regions , double and triple, single and double stranded RNA and RNA which is a mixture of single and double stranded regions, hybrid molecules comprising DNA and RNA which may be single stranded or, more typically, double stranded or triple stranded or a mixture of single and double chain regions. In addition, the term "polynucleotide", as used herein, refers to triple chain regions comprising RNA or DNA or both RNA and DNA. The chains of these regions can come from the same molecule or from different molecules. The regions can include everything from one or more of the molecules, but more typically they only comprise a region of some of the molecules. One of the molecules of a triple helical region is often an oligonucleotide. As used herein, the term "polynucleotide" includes the previously described DNA or RNA containing one or more modified bases. Therefore, DNA or RNA with modified parent structures for greater stability or for other reasons are "polynucleotides" according to the meaning of said term herein. Moreover, DNA or RNA comprising uncommon bases, such as inosine, or modified bases, such as tritylated bases, to name but two examples, are polynucleotides according to the meaning of said term herein. It will be understood that numerous and varied modifications have been made to DNA and RNA that serve many of the purposes known to those skilled in the art. The term polynucleotide, as used herein, encompasses such chemically, enzymatically or metabolically modified forms of the polynucleotides, as well as the chemical forms of the DNA and RNA characteristic of viruses and cells, including, inter alia, simple and complex cells. POLYPEPTIDES, as used herein, include all polypeptides that will be described later. The basic structure of the polypeptides is well known and is described in countless textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined together in a linear chain by peptide bonds. As used herein, the term refers to both short chains, also commonly known in the art as peptides, oligopeptides and oligomers, for example, as to longer chains, which are generally known in the art as proteins, of which there are many types. It will be understood that polypeptides often contain amino acids other than the 20 amino acids commonly known as the 20 natural amino acids, and that many amino acids, including terminal amino acids, can be modified in a given polypeptide, not only by natural processes, such as processing and other post-translation modifications, but also by chemical modification techniques that are well known to the art. Even the common modifications that occur naturally in the polypeptides are too numerous to make an exhaustive enumeration in the present, but they are very well described in the basic texts and in more detailed monographs, as well as in the voluminous research literature, and they are well known by art specialists. Among the known modifications that can be found in the polypeptides of the present invention are, to name a few, by way of illustration, acetylation, acylation, ADP-ribosylation, amidation, covalent binding of flavin, covalent attachment of a heme group, covalent of a nucleotide or derivative of a nucleotide, covalent attachment of a lipid or a derivative of a lipid, covalent binding of phosphatidylinositol, crosslinking, delation, formation of disulfide bonds, demethylation, formation of covalent crosslinks, cystine formation, pyroglutamate formation , formylation, gamma-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid addition mediated by RNA transfer to proteins such as arginylation and ubiquitination. These modifications are well known to the specialist and are described in greater detail in the scientific literature. Several of the particularly common modifications, such as for example glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, are described in most of the basic texts, such as, for example, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2a Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). There are many reviews available on this subject, such as, for example, those provided by Wold, F., Post-translational Protein Amendments: Perspectives and Prospects, p. 1-12 in POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. O Johnson, Ed., Academic Press, New York (1983); Seifter eí al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). It will be understood, as is known and previously indicated, that the polypeptides are not entirely linear. For example, polypeptides can be branched as a result of ubiquitination and can be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events due to human manipulation that do not take place naturally. Circular, branched and circular branched polypeptides can be synthesized by a natural non-translation process and also by methods that are only synthetic. The modifications can take place at any location in a polypeptide, including in the main structure of the peptide, in the side chains of the amino acids and in the terminal amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in natural and synthetic polypeptides and such modifications can also be found in the polypeptides of the present invention. For example, the amino terminal residue of the polypeptides obtained in E. coli or other cells, before proteolytic processing, will almost invariably be N-formylmethionine. During the post-translational modification of the peptide, a methionine residue may be deleted at the NH2- terminus terminal. Accordingly, this invention contemplates the use of methionine-containing variants and variants that do not contain amino-terminal methionine of the protein of the invention. The modifications that take place in a polypeptide will often be a function of how it is effected. For polypeptides made by expression of a gene cloned in a host, for example, the nature and extent of the modifications will be largely determined by the ability of post-translational modification of the host cell and the modification signals present in the host cell. amino acid sequence of the polypeptide. For example, as is well known, glycosylation often does not occur in bacterial hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, the polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Similar considerations apply to other modifications. It will be understood that the same type of modification may be present with the same degree or in varying degrees at several sites in a given polypeptide. In addition, a given polypeptide can contain many types of modifications. In general, as used herein, the term "polypeptide" encompasses all such modifications, particularly those that are present in polypeptides synthesized by expression of a polynucleotide in a host cell. PROMOTER, as used herein, includes any reference to a region of DNA located d 'with respect to the initiation of transcription and is engaged in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Examples of plant promoters include, for example, those obtained from plants, viruses and plant bacteria comprising genes expressed in plant cells, such as Agrobacterium or Rhizobium. Examples of promoters under development control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, or seeds or spatially in regions, such as endosperm, embryos or meristematic regions. Said promoters are known as "preferably tissue" promoters. Promoters that initiate transcription only in certain tissues are termed "tissue-specific". A temporarily regulated promoter directs expression at particular times, such as between 0-2d days after pollination. A promoter "with preference for a cell type" directs primarily the expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter that is under development control and may be inducible or liable to derepression. Examples of environmental conditions that can effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-specific, cell-type and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active under most environmental conditions and in all or almost all tissues, in all or almost all stages of development. RECOMBINANT EXPRESSION CYSET, as used herein, refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specific genetic elements that allow the transcription of a particular nucleic acid in a cell Guest. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the portion of the recombinant expression cassette of an expression vector includes, among other sequences, the nucleic acid to be transcribed and a promoter, and optionally may comprise additional elements, such as an enhancer. RELATED FEMALE REPRODUCTIVE TISSUE, as used herein, includes maternal plant tissues, such as female florets, ovaries, aleurone, pedicels, and pedicel-forming regions, either before or after pollination. Seed pre-pollination tissues may also be referred to as "grain starters" or "seed starters". TRANSFORMATION, as used herein, is the process by which a cell is "transformed" with an exogenous DNA when said exogenous DNA has been introduced into the cell membrane. The exogenous DNA can be integrated, or not, (covalently linked) into the chromosomal DNA that makes up the genome of the cell. In prokaryotes and yeasts, for example, the exogenous DNA can be maintained on an episomal element, such as a plasmid. With respect to higher eukaryotic cells, a stably transformed or transfected cell is one in which the exogenous DNA has been integrated into the chromosome in such a way that it is inherited by the daughter cells through the replication of the chromosomes. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones composed of a population of daughter cells containing the exogenous DNA.

VARIANT (S) of polynucleotides or polypeptides, as such term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. These types of variants are described below and in another part of the present description in greater detail. With reference to the polynucleotides, in general, the differences are limited such that the nucleotide sequences of the reference and of the variant are generally similar in general and, in many regions, identical. As will be indicated below, changes in the nucleotide sequence of the variant may be silent; that is, they possibly do not alter the amino acids encoded by the polynucleotide. When the alterations are limited to silent changes of this type, the variant will encode a polypeptide with the same amino acid sequence as the reference. In other cases, as will be indicated below, changes in the nucleotide sequence of the variant can alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Said nucleotide changes may result in one or more amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by said reference sequence, as will be described below. With reference to the polypeptide variants in general, the differences are limited so that said sequences of the reference and of the variant are closely similar in general and, in many regions, are identical. A polypeptide variant and polypeptide reference may differ in amino acid sequence in one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. VIGOR of a plant, as used herein, refers to the relative health, productivity and growth rate of the plant and / or of certain parts of the plants, and can be seen reflected in many development attributes, including, for example, chlorophyll concentration, photosynthesis index, total biomass, root biomass , quality of the grains and / or yield of the grains. In Zea mays in particular, vigor may also be reflected in the growth rate of ears of corn, size of ears and / or ability to expand styles. The vigor can be determined with reference to different genotypes under similar environmental conditions or with reference to the same genotype or different genotypes under different environmental conditions. STABILITY OF PERFORMANCE, as known in the art and as used herein, refers to the consistent yield of a given genotype in different environments, including stress environments.

DETAILED DESCRIPTION OF THE INVENTION: This invention relates, in part, to nucleic acid constructs useful for modulating cytokinin activity in plants, including temporal and / or spatial expression of cytokinin genes in seeds and related female reproductive tissue, and with associated polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and polypeptides, and the variants and derivatives thereof; polypeptide agonists and antagonists; products comprising these polynucleotides and polypeptides and the variants and derivatives thereof; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists and uses of the products comprising them. In particular, in this and in other respects, the invention relates to polynucleotides and polypeptides of the metabolic pathway of cytokinins, including the enzymes ipt and cytokinin oxidase and the genes encoding them and their use alone or in combination with each other and / or in combinations with various other isolated polynucleotides and polypeptides that affect the activity of cytokinins. The directed modulation of the expression is described to increase the vigor of the plants and the yield of the seeds. As mentioned above, the invention provides the necessary reagents for the development of transgenic plants characterized by increased cytokinin activity. As used herein, the phrase "cytokinin activity" is relative and refers to cytokinin activity in a control plant without transgenes that affects the cytokinins compared to a plant with said functional transgene. Relative levels can also be measured using only the transgenic plant but measured in presence and absence of expression of the transgene of interest. Accordingly, any structural gene, whose regulated expression has the effect of increasing the activity of cytokinins in plants, in particular seeds, is useful for the practice of this invention. Genes that direct the expression of proteins that act by increasing the biosynthesis of cytokinins (e.g., ipt or tzs) or the genes encoding cytokinin degrading enzymes, whose expression is inhibited, can be used in the practice of this invention. However, this invention also contemplates the use of other genes. In addition to genes that affect the absolute levels of cytokinins, genes that affect the ratio of cytokinins to auxins are also useful. Genes that decrease auxin levels, such as iaa-1, and gene-5 can also be employed in the practice of this invention. In addition, or alternatively, a directed modulation of the expression of isolated polynucleotides encoding the polypeptides involved in recognition and cellular response to cytokinins may provide enhanced cytokinin activity, as defined herein. Also contemplated are combinations of these approaches, comprising changes in the expression of one or more cytokinin modulator genes. As mentioned above, the present invention relates to novel polypeptide constructs of the metabolism of cytokinins and to the polynucleotides encoding them, among others, as will be described in more detail below. Polypeptides of particular utility for the practice of this invention include, for example, ipt and cytokinin oxidase. The nucleic acids, and fragments thereof, which encode the mentioned enzymes of utility to generate transgenic producers of enzymes. By example, a single gene or gene fragment (or combinations of several genes) can be incorporated into an appropriate expression cassette (using, for example, the globulin-1 [glbl] promoter for a preferred expression in embryos, or the 27 kd gamma-zein promoter for a preferred expression in the endosperm of the seeds) and transform it into corn together with an appropriate selectable marker (such as the BAR and PAT genes). Certain embodiments comprise a promoter that directs expression in female reproductive meristematic tissue operably linked to a polynucleotide encoding a cytokinin biosynthesis enzyme. Examples of promoters that are useful in such embodiment include zag2.1, Zap (also known as ZmMADS), tb1 and PCNA2, as shown in SEQ ID N °: 3, d, 17 and 2d. In certain situations it may be preferable to silence or subsensitize certain genes, such as cytokinin oxidase. The most important literature describing the application of genes for silencing dependent on homology include: Jorgensen, Trends Biotechnol. 8 (12): 340-344 (1990); Flavell, Proc. Nati Acad. Sci. (USA) 91: 3490-3496 (1994); Finnegan et al., Bio / Technology 12: 883-888 (1994); Neuhuber went to., Mol. Gen. Genet. 244: 230-241 (1994); Flavell et al. (1994) Proc. Nati Acad. Sci. USA 91: 3490-3496; Jorgensen e to al. (1996) Plant Mol. Biol. 31: 9d7-973; Johansen and Carrington (2001) Plant Physiol. 126: 930-938; Broin I went to. (2002) Plant Cell 14: 1417-1432; Stoutjesdijk I went to. (2002) Plant Physiol. 129: 1723-1731; Yu I went to. (2003) Phytochemistry 63: 7d3-763; and U.S. Pat. N °: d.034.323, 5.283.184 and 5.942.657. Alternatively, another approach to silencing genes comprising antisense technology can be used (Rothstein et al.

Plant Mol. Cell. Biol. 6: 221-246 (1989); Liu ei al. (2002) Plant Physiol. 129: 1732-1743 and U.S. Pat. N °: 5,759,829 and 5,942,657. Methods and constructs for subsensitizing the expression of cytokinin oxidase are described in U.S. Patent Application Ser. Co-pending, Cytokinine Oxidase-Like Sequences and Methods of Use, 60 /, presented on April 2, 2004. Certain embodiments may comprise both enhanced cytokinin biosynthesis and reduced degradation of cytokinins to result in increased cytokinin activity. Polynucleotides According to one aspect of the present invention, there are provided the isolated polynucleotides of SEQ ID N °: 26, 28, 30 and 32, which encode the enzymes of the metabolism of maize cytokinins, cytokinin oxidases, which possess the sequences of deduced amino acids shown here as SEQ ID N °: 27, 29, 31 and 33, described in the Copending Provisional Application, Citokinine Oxidase-Like Sequences and Methods of Use, 60 /, presented on April 2, 2004; as well as the corn cytokinin oxidase of SEQ ID N °: 38, which encodes SEQ ID N °: 39, described in U.S. Pat. No. 6,229,066 and in WO99 / 06571. The use of the isolated polynucleotide encoding ipt (isopentenyl transferase), provided in Molecular and General Genetics 216: 388-394 (1989) and provided herein as SEQ ID NO: 1, and its deduced amino acid sequence of SEQ ID N °: 2, are also contemplated by this invention, as well as the use of other cytokinin biosynthesis genes (eg, ipt) isolated from other organisms, such as, for example, Arabidopsis or corn.

In accordance with one aspect of the present invention, the polynucleotide isolated from Agrobacterium tumefaciens encoding isopentenyl transferase, SEQ ID NO: 1, and its deduced amino acid sequence, SEQ ID NO: 2 (Strabala, et al., Mol. Gen. Genet, 216, 388-394 (1989); GenBank, Accession No. X14410); the corn promoter Zag2.1, SEQ ID N °: 3 (GenBank X80206); the CaMV 3ds enhancer, SEQ ID N °: 4; the corn Zap promoter, SEQ ID N °: 5 (also known as ZmMADS; U.S. Patent Application No. 10 / 387,937; WO 03/078590); the corn ckx1-2 promoter, SEQ ID N °: 6 (U.S. Patent Publication No. 2002-0162500 A1, WO 02/0078438); the corn eepl promoter, SEQ ID N °: 7 (U.S. Provisional Application No. 60 / 460,718); the corn end2 promoter, SEQ ID N °: 8 (U.S. Patent Applications No.:6d28,704 and 10 / 310,191); the corn led promoter, SEQ ID N °: 9 (U.S. Patent Application No.: 09 / 718.7d4); the corn F3.7 promoter, SEQ ID NO: 10 (Baszczynski et al., Maydica 42: 189-201 (1997); the corn tb1 promoter, SEQ ID N °: 17 (Hubbarda et al., Genetics 162 : 1927-193d, December 2002), the corn eep2 promoter, SEQ ID N °: 18, the corn thioredoxin H promoter, SEQ ID N °: 19, U.S. Provisional Application N °: 60 / 614,123); the corn promoter Zm40, SEQ ID NO: 20 (U.S. Patent No. 6,403,862 and WO 01/2178); the corn mLIP1d promoter, SEQ ID N °: 23 (U.S. Patent No.: 6,479,734); the corn ESR promoter, SEQ ID No. 24 (U.S. Patent Application No. 10 / 786,679, filed February 2, 2004); the corn PCNA2 promoter, SEQ ID N °: 25 (U.S. Patent Application Serial No. 10 / 388,359, filed March 13, 2003); cytokinin oxidases and corn promoters, SEQ ID N °: 26-37 (Copending Provisional Application, Citokinine Oxidase-Like Sequences and Methods of Use, 60 /, presented on April 2, 2004). The corn ZAG2 gene was isolated on the basis of homology with the AGAMOUS gene of Arabidopsis, which directs floral development. (Schmidt et al., Plant Cell 5 (7): 729-737, 1993). Normally, the ZAG2 gene is expressed primarily in developing female florets. The coding sequence of ZAG2 and approximately 2.1 kb of 5 'sequence were deposited GenBank with Accession No. X80206 in September 1995. A portion of the d' region of ZAG2 is included here as SEQ ID NO: 3 and is referred to as ZAG2.1 promoter. The use of the information provided here, such as the polynucleotide sequences that will be described below, allows to obtain a polynucleotide of the present invention that encodes polypeptides of cytokinin metabolism enzymes using standard cloning and searching procedures. To obtain the polynucleotide encoding the protein using the DNA sequences described below, oligonucleotide primers that are complementary to the known polynucleotide sequence can be synthesized. These primers can then be used in a PCR to amplify the polynucleotide from a temperate derivative of mRNA or genomic DNA isolated from the desired source. The resulting amplified products can then be cloned into commercial cloning vectors, such as the TA vector series of InVitrogen. The sequencing of the individual clones thus identified with sequencing primers designed from the original sequence allows the sequence to be extended in both directions in order to determine the complete genetic sequence. Said sequencing is carried out using denatured double-stranded DNA prepared from a plasmid clone. The suitable techniques were described by Maniatis, T., Fritsch, E.F. and Sambrook, J. in MOLECULAR CLONING, A Laboratory Manual (2nd edition, 1989, Cold Spring Harbor Laboratory, See, Sequencing Denatured Double-Stranded DNA Templates, 13.70 Isolation of the ipt gene The isopentenyl transferases (ipt) of the present invention are can be obtained from sources including, for example, Zea mays, Agrobacterium, Psuedomonas savastano, Rhodococcus and Erwinia The complete sequence of the ipt gene is provided in Strabala, TJ, et al., Isolation and Characterization of an ipt gene from the Ti plasmid Bo542, Mol. Gen. Genet 216, 388-94 (1989) .A copy of said gene can be prepared by synthesis using DNA synthesis protocols well known to those skilled in the art of gene synthesis. Alternatively, a copy of the gene can be isolated directly from an organism containing the ipt gene, for example by PCR cloning as described in WO 00/63401, incorporated herein by reference. The polynucleotides of the present invention can be found in the form of RNA, such as mRNA, or in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or produced by chemical synthesis techniques or by a combination of the same. The DNA can be double-stranded or single-stranded. The single-stranded DNA can be the coding strand, also known as the strand oriented in the sense of reading, or it can be a non-coding strand, also known as an antisense strand. The coding sequence encoding the polypeptide can be identical to the coding sequence of the polynucleotides shown below. It can also be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptides shown below. As will be described in more detail below, these alternative coding sequences constitute an important source of sequences for codon optimization. The polynucleotides of the present invention that encode the polypeptides listed below include, for example, the coding sequence corresponding to the mature polypeptide, per se; the coding sequence corresponding to the mature polypeptide and additional coding sequences, such as those encoding direct or secretory sequences, such as the sequence of a pre- or pro- or prepro-protein; the coding sequence of the mature polypeptide, with or without the additional coding sequences mentioned, together with other additional non-coding sequences, including for example, without limitations, non-coding sequences d 'and 3', such as the transcribed, untranslated sequences which fulfill functions in transcription (including, for example, termination signals), ribosome binding, mRNA stability elements, and other coding sequences encoding additional amino acids, such as those that provide additional functionalities. The DNA may also comprise promoter regions that direct transcription of the DNA encoding the cytokinin-modulating heterologous enzymes of this invention. The term heterologous is defined as a sequence that is not natural with the promoter sequence. Although the nucleotide sequence is heterologous for the promoter sequence, it can be homologous (native) or heterologous (strange) for the plant host. Still further, the polypeptide can be fused to a marker sequence, such as a peptide, which facilitates the purification of the fused polypeptide. In certain embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc.) and the pET vector series (Novagen), among others, many of which are commercially available. As described in Gentz et al., Proc. Nati Acad. Sci., (USA) 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. The HA tag may also be used to create fusion proteins and corresponds to an epitope derived, for example, from the influenza hemagglutinin protein, which was described by Wilson et al., Cell 37: 767 (1984). According to the aforementioned, the term "polynucleotide encoding a polypeptide", as used herein, encompasses polynucleotides that include a coding sequence for a polypeptide of the present invention, in particular cytokinin modulating enzymes that display the sequences of amino acids shown below. The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (eg, interrupted by an integrated phage or an insertion or editing sequence) together with additional regions that may also contain coding and / or non-coding sequences. The present invention further relates to variants of the present polynucleotides encoding fragments, analogs and derivatives of the polypeptides having the amino acid sequence deduced below. A variant of the polynucleotide can be a natural variant, such as a natural allelic variant, or it may be a variant that does not appear naturally. Such unnatural variants of the polynucleotide can be obtained by mutagenesis techniques, including those that can be applied to polynucleotides, cells or organisms. Variants of this type include variants that differ from the polynucleotides mentioned by substitutions, deletions or additions of nucleotides. The substitutions may comprise one or more nucleotides. The variants can be altered in the coding or non-coding regions or in both. Alterations in the coding regions can produce substitutions, deletions or additions of conservative or non-conservative amino acids. Among the embodiments of the invention of this type, there are polynucleotides encoding polypeptides having the amino acid sequences shown below; variants, analogs, derivatives and fragments thereof. In addition, in this sense, there are polynucleotides that encode variants, analogs, derivatives and fragments of cytokinin biosynthesis enzymes, and variants, analogs and derivatives of said fragments, which have the amino acid sequences shown below in which replaced, deleted or added several, a few, 1 to 10, 1 ad, 1 to 3, 2, 1 or no amino acid residues, in any combination. Among them are polynucleotides that comprise substitutions, additions and silent deletions, which do not alter the properties and activities of the enzymes of cytokinin biosynthesis; conservative substitutions; and polynucleotides encoding polypeptides having the amino acid sequence that is show later, without substitutions. Other embodiments of the invention comprise polynucleotides that are greater than 79%, at least 80% or at least 8d% identical to a polynucleotide that encodes a polypeptide having the amino acid sequence shown below, and polynucleotides that are complementary to said polynucleotides. Furthermore, certain embodiments comprise polynucleotides encoding polypeptides that retain substantially the same function or biological activity, or even exhibit an increase thereof, as compared to that of the mature polypeptide encoded by the polynucleotides shown below. The present invention also relates to polynucleotides that hybridize with the sequences described above. In this sense, the present invention relates especially to polynucleotides that hybridize under severe conditions with the polynucleotides described above. As used herein, the term "severe conditions" means that hybridization will only occur if there is at least 80% identity between the sequences. The terms "severe conditions" or "severe hybridization conditions" refer to conditions under which a probe will hybridize to its target sequence, to a greater detectable extent than to other sequences (e.g., at least 2 times the level). basal). Severe conditions depend on the sequence and will be different under different circumstances. The control of the severity of the hybridization and / or washing conditions allows to identify white sequences that are 100% complementary to the probe (probe homologs). As an alternative, it is it is possible to adjust the severity conditions to allow some mismatch in the sequences so that lower degrees of similarity are detected (heterologous probes). In general, a probe is less than about 1000 nucleotides in length, often less than 500 nucleotides in length. Typically, severe conditions will be those in which the concentration of salts is less than about 1.5 M of Na, typically between 0.01 and 1.0 M approximately Na (or other salts) concentration. at pH between 7, 0 and 8.3 and the temperature is at least about 30 ° C for short probes (for example, between 10 and 50 nucleotides) and at least about 60 ° C for long probes (for example, more than dO nucleotides). Severe conditions can also be achieved with the addition of destabilizing agents, such as formamide. Examples of low stringency conditions include hybridization with 30 to 3d% formamide buffer, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C and 1X to 2X SSC wash (SSC 20X = NaCl 3.0 M / 0.3 M trisodium citrate) at dO-dd ° C. Examples of moderate severity conditions include hybridization in formamide 40 to 45%, NaCl 1M, SDS 1% at 37 ° C and a wash in SSC 0.5X to 1X at dd-60 ° C. Examples of conditions of high severity include hybridization in 60% formamide, 1 M NaCl, 1% SDS at 37 ° C and a wash in 0.1X SSC at 60-6d ° C. The specificity typically depends on the post-hybridization washes, the critical factors being the ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Tm = 81, 6 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - dOO / L; where M is the molarity of the monovalent cations,% GC is the percentage of guanosine and nucleotides of cytosine in the DNA,% form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 60% of a complementary white sequence is hybridized with a perfectly matching probe. The Tm is reduced by approximately 1 ° C for every 1% of mismatch; therefore, it is possible to adjust the Tm, the hybridization and / or washing conditions to hybridize the sequences of the desired identity. For example, if you search for sequences with > 90% identity, the Tm can be decreased by 10 ° C. In general, severe conditions are selected to be approximately d ° C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, very severe conditions may employ hybridization and / or a wash at 1, 2, 3 or 4 ° C less than the thermal melting point (Tm); moderately severe conditions may utilize hybridization and / or a wash at 6, 7, 8, 9 or 10 ° C less than the thermal melting point (Tm); Low stringency conditions can employ hybridization and / or washing at 11, 12, 13, 14, 15 or 20 ° C less than the thermal melting point (Tm). Using the equation, the hybridization and washing compositions and the desired Tm, the skilled artisan will understand that variations in the severity of the hybridization and / or wash solutions are inherently described. If the degree of mismatch desired results in a Tm less than 45 ° C (aqueous solution) or 32 ° C (formamide solution) it is preferred to increase the concentration of SSC so that a temperature can be used highest. Hybridization and / or washing conditions can be applied for at least 10, 30, 60, 90, 120 or 240 minutes. A very extensive guide for nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acids Probes, Part I, Chapter 2"Overview of Principles of Hybridation and the Strategy of Nucleic Acids Probes Assays" , Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). As already mentioned herein with respect to the polynucleotide assays of the invention, for example, the polynucleotides of the invention can be used as hybridization probes for RNA, cDNA and genomic DNA in order to isolate full-length cDNA and clones genomic encoders of enzymes of cytokinin biosynthesis and to isolate cDNA and genomic clones of other genes that have a high sequence similarity with said genes. Said probes will generally comprise between 15 and 60 bases approximately. The polynucleotides and polypeptides of the present invention can be used as reagents and research materials to discover transgenic plants with a modulated cytokinin activity. The polynucleotides of the invention that are oligonucleotides derived from the sequences described below can be used as primers for PCR in the process described herein to determine whether the genes identified herein are transcribed, or not, in whole or in part in the tissue that accumulates cytokinins. The polynucleotides can encode a polypeptide that is the protein matures more additional amino or carboxyl-terminal amino acids or inner amino acids of the mature polypeptide (eg, when the mature form has more than one polypeptide chain). Said sequences can fulfill a function in the processing of the protein from a precursor to a mature form, they can allow the transport of proteins, they can prolong or shorten the half-life of the protein or they can facilitate the manipulation of the protein for tests or for production, among other things. As is generally the case in vivo, the additional amino acids can be processed for their separation from the mature protein by cellular enzymes. A precursor protein, where the mature form of the polypeptide is fused to one or more prosequences, may be an inactive form of the polypeptide. When the prosequences are separated, said precursors are generally activated. Some or all of the prosequences may be separated before their activation. In general, said precursors are called proproteins. In summary, a polynucleotide of the present invention can encode a mature protein, a mature protein plus a leader sequence (which can be termed a preprotein), a precursor of a mature protein containing one or more prosequences that do not constitute direct sequences of a preprotein , or a preproprotein that is a precursor of a proprotein, which contains a leader sequence and one or more prosequences, which are generally separated during the processing steps that produce the active and mature forms of the polypeptide. Polypeptides The present invention also relates to polypeptides that they comprise the amino acid sequences deduced below. A polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain embodiments it is a recombinant polypeptide. The invention also relates to fragments, analogs and derivatives of these polypeptides. The terms "fragment", "derivative" and "analogue", with reference to the polypeptides, refer to a polypeptide that retains at least 90% or at least 95% or essentially the same function or biological activity of said polypeptide . Therefore, an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Among the embodiments of the invention of this type, there are polypeptides having the amino acid sequence of cytokinin modulating enzymes shown below, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of said fragments A fragment, derivative or analog of the polypeptides shown below can be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and said conserved amino acid residue may be encoded, or not, by the genetic code or (ii) one in which one or more of the amino acid residues includes a substituent group or (iii) one in which the mature polypeptide is fused with another compound, such as a compound for increasing the half-life of the polypeptide (eg, polyethylene glycol) or (iv) one in which additional amino acids are fused to the mature polypeptide, such as a sequence guideline or secretory or a sequence used to purify the mature polypeptide or a sequence of a proprotein. It is considered that those skilled in the art can obtain said fragments, derivatives and the like from the explanations proposed herein. Among the preferred variants are those that vary with respect to the reference by conservative amino acid substitutions. Said substitutions are those that substitute a given amino acid in a polypeptide for another amino acid of similar characteristics. Typically, replacements, one for another, of the aliphatic amino acids Ala, Val, Leu and He are considered as conservative substitutions; exchange of the hydroxyl residues Ser and Thr, exchange of the acid residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg; and replacements between the aromatic residues Phe, Tyr. Furthermore, variants, analogs, derivatives and fragments, and variants, analogs and derivatives of said fragments, which have the amino acid sequences shown below, in which several, have been substituted, deleted or added are particularly preferred in this regard. , a few, 1 to 10, 1 ad, 1 to 3, 2, 1 or no amino acid residue, in any combination. Among them, substitutions, additions and silent deletions, which do not alter the properties and activities of the enzymes of cytokinin biosynthesis, are especially preferred. In this sense, conservative substitutions are also especially preferred. Even more preferred are polypeptides having the amino acid sequences shown below without substitutions. The polypeptides and polynucleotides of the present invention are provided preferably in an isolated form and can be purified to homogeneity. Vectors, host cells, expression The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells that have incorporated the vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Vectors According to this aspect of the invention, the vector can be, for example, a vector of a plasmid, a vector of a single or double chain phage, a viral vector of RNA or single or double-stranded DNA. Such vectors can be introduced into cells as polynucleotides, preferably DNA, by well-known techniques for introducing DNA and RNA into cells. Vectors, in the case of phage and viral vectors, can also be introduced, and preferably, into cells in as packaged or encapsulated viruses by well-known infection and transduction techniques. Viral vectors may be replication competent or defective for replication. In the latter case, viral propagation will generally take place in complementary host cells. Among the vectors, those which are for the expression of polynucleotides and polypeptides of the present invention are preferred in certain senses. In general, said vectors comprise control regions that act in cis effective for expression in a host, operatively linked to the polynucleotide to be expressed. The appropriate factors that act in trans are supplied by the host, supplied by a complementary vector or supplied by the vector itself after its introduction into the host. In certain preferred embodiments of this type, the vectors provide a preferred expression. Said preferred expression may be an inducible or temporarily limited or predominantly restricted expression to certain cell types or any combination of the above-mentioned options. Among the particularly preferred inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable for this aspect of the invention exist, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, which are well known to those skilled in the art and which employ them routinely. Such vectors include, inter alia, chromosomal, episomal and virus derivatives, for example, vectors derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as such as baculovirus, papova virus, such as SV40, vaccinia virus, adenovirus, bird pox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids and binaries used for transformations mediated by Agrobacterium. All can be used for the expression according to this aspect of the present invention. The following vectors, commercially available, are only offered by way of example. Preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, vectors Fagoscript, Bluescript vectors, vectors pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDRd40, pRITd available from Pharmacia. Preferred vectors for eukaryotes are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. The plant binary vectors that are useful include BIN19 and its available Clontech derivatives. These vectors are listed only by way of illustration of the numerous commercial and well-known vectors that can be used by those skilled in the art for use in accordance with this aspect of the present invention. It will be understood that it is possible to use any other suitable plasmid or vector, for example, for the introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host in this aspect of the invention, several of which are described with more detail later. In general, expression constructs will contain sites to initiate and terminate transcription and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include an AUG translation initiation codon at the beginning and a termination codon appropriately located at the end of the polypeptide to be translated. In addition, constructions may contain control regions that regulate as well as generate expression. In general, according to many procedures that are of common practice, said regions will function controlling transcription, such as transcription factors, binding repressor sites and termination signals, among others. For the secretion of the translated protein towards the lumen of the endoplasmic reticulum, towards space periplasmic or into the extracellular environment, it is possible to incorporate appropriate secretion signals into the expressed polypeptide. These signals may be endogenous to the polypeptide or may be heterologous signals. The transcription of the DNA encoding the polypeptides of the present invention can be increased by higher eukaryotes by inserting an enhancer sequence into the vector. Enhancers are elements of DNA that act in cis, usually from about 10 to 300 bp which act by increasing the transcription activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the late side of the replication origin, and enhancers of adenovirus. Other enhancers that are useful in the invention for increasing the transcription of the introduced DNA segment include, among others, viral enhancers such as those found within the 3dS promoter described by Odell et al., Plant Mol. Bioi. 10: 263-72 (1988), and an opin gene enhancer described by Fromm et al., Plant Cell 1: 977 (1989). The enhancer may affect the tissue specificity and / or the temporal specificity of the expression of the sequences included in the vector. For example, the construct may comprise the CaMV 3ds enhancer (SEQ ID N °: 4) oriented "head to head" with respect to the zag2.1 promoter (SEQ ID NO: 3) which directs the ipt gene (SEQ ID NO. : 1). The termination regions also facilitate efficient expression by terminating transcription at the appropriate points. Terminators that are useful for the practice of this invention include, for example, pinll (See An et al., Plant Cell 1 (1): 116-122 (1989)), glbl (See GenBank, Accession No. L22346), gz (See terminator gzw64a, GenBank, Accession No. S78780) and terminator nos Agrobacterium Known eukaryotic promoters that are suitable for generalized expression are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the retroviral LTR promoters, such as Rous sarcoma virus. ("RSV"), metallothionein promoters, such as the mouse metallothionein-l promoter and various plant promoters, such as globulin-1. When available, the native promoters of the cytokinin modulator enzyme genes can be used. Those that are representative of prokaryotic promoters include the lambda PL phage promoter, the lac, trp and tac promoters of E. coli to name a few of these well-known promoters. With respect to plants, examples of promoters with preference for seeds include promoters of seed storage proteins that express these proteins in seeds in a highly regulated manner (Thompson, et al., BioEssays;. 10: 108 (1989) ), such as, for dicotyledonous plants, the β-phaseolin promoter of bean, the napin promoter, the β-conglycinin promoter and the soybean lectin promoter. For monocotyledonous plants, promoters that are useful for the practice of the invention include, for example, in maize the 15 kD zein promoter, the 22 kD zein promoter, the 27 kd? -zein promoter (such as the gzw64A promoter, see GenBank, Accession No. S78780), the waxy promoter, the shrunken promoter - "\, the globulin 1 promoter (See GenBank, Accession No. L22344), the Itp2 promoter (Kalla, et al. ., Plant Journal 6: 849-860 (1994); U.S. Patent No. 6,626,716), the ciml promoter (see U.S. Patent No. 6,225,529), the corn endl and end2 promoters (See U.S. Patent No. 6,528. 704 and Application 10 / 310,191, filed December 4, 2002); the nuci promoter (U.S. Patent No.: 6,407,315); the Zm40 promoter (U.S. Patent No. 6,403,862); eepl (SEQ ID N °: 7) and eep2 (SEQ ID N °: 18); led (U.S. Patent Application No.: 09 / 718,754); the thioredoxin H promoter (U.S. Provisional Application Number: 60 / 514,123;; the mlip15 promoter (U.S. Patent No.: 6,479,734); the PCNA2 promoter, SEQ ID N °: 25, and the shrunken-2 promoter (Shaw et al., Plant Phys 98: 1214-1216, 1992; Zhong Chen et al., PNAS USA 100: 3526-3530, 2003). in the art they also know other promoters that are useful in the practice of the invention, such as the nucellain promoter (See O Linnestad, et al., Nucellain, A Barley Homolog of the Dichotic Vacuolar - Processing Protease Is Localized in Nucellar Cell Walls, Plant Physiol. 118: 1169-80 (1998), the kn1 promoter (See S. Hake and N. Ori, The Role of knottedl in Meristem Functions, B8: INTERACTIONS AND INTERSECTIONS IN PLANT PATHWAYS, COEUR D'ALENE, IDAHO, KEYSTONE SYMPOSIA, February 8-14, 1999, in 27.) and the promoter F3.7 (Baszczynski et al., Maydica 42: 189-201 (1997), SEQ ID N °: 10). spatial action, such as glbl , a promoter with preference for embryos; or of gamma zein, a promoter with preference for the endosperm; or an active promoter in the region surrounding the embryo (see U.S. Patent Application No. 10 / 786,679, filed February 2, 2004) or BETL1 (See G. Hueros, et al., Plant Physiology 121: 1143-1162 (1999) and Plant Cell 7: 747-57 (June 1995)), are of particular utility, including promoters that are preferentially active in female reproductive tissues, and those that are active in meristematic tissues, in particular in female reproductive meristematic tissues. The use of temporary action promoters is also contemplated by this invention. Promoters that act on days 0-25 after pollination (DAP) are preferred, as well as those who act 4-21, 4-12 or 8-12 DAP. In this regard, promoters such as ciml and Itp2 are preferred. Promoters that act -14 to 0 days after pollination, such as SAG12 (See WO 96/29858, Richard M. Amasino, published October 3, 1996) and ZAG1 or ZAG2 (See RJ Schmidt, may also be used. et al., Identification and Molecular Characterization of ZAG1, the Maize Homolog of the Arabidopsis Floral Homeotic Gene AGAMOUS, Plant-Cell 5 (7): 729-37 (July 1993) See also SEQ ID N °: 3). Promoters that are useful include corn zag2.1 (SEQ ID N °: 3), Zap (SEQ ID N °: 5, also known as ZmMADS); Application U.S. Patent No.: 10 / 387,937; WO 03/078690); the corn tb1 promoter (SEQ ID N °: 17; see also Hubbarda et al., Genetics 162: 1927-1936, 2002).

Examples of suitable promoters for a generalized expression in plants are the promoter of the small subunit of ribulose-1, d-bi-phosphate carboxylase, the promoters of the tumor-inducing plasmids of Agrobacterium tumefaciens, such as the nopaline promoters. synthetase and octopine synthetase, and viral promoters, such as the 19S and 35S promoters of the cauliflower mosaic virus (CaMV) or the 35S promoter of the scrofularia mosaic virus. It must be borne in mind that there are numerous promoters who do not mentioned and are suitable for use in this aspect of the invention, which are also well known and can be readily employed by the specialists in the manner illustrated in this description and the examples herein. For example, this invention contemplates the use, where appropriate, of the native promoters of the cytokinin biosynthesis enzymes to direct the expression of the enzyme in a recombinant environment. The propagation and expression vectors will generally include selectable markers. Said markers may also be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for the selection of transformed host cells. Preferred markers include dihydrofolate reductase genes or neomycin resistance for the culture of eukaryotic cells and tetracycline or ampicillin resistance genes for the culture of E. coli and other prokaryotes. In plant systems, resistance genes are generally useful. to kanamycin and herbicides (PAT and BAR). Genes of selectable markers, physically attached to the introduced DNA segment, are used to recover the transformed cells either by positive genetic selection or search and examination. The genes of selectable markers also allow maintaining a selection pressure on a population of transgenic plants, to ensure that the introduced DNA segment, and its respective promoters and control enhancers, are retained by the transgenic plant. Many of the genes of positive selectable markers used Commonly for the transformation of plants they were isolated from bacteria and they encode enzymes that metabolically detoxify a selective chemical agent that can be an antibiotic or a herbicide. Other positive selection marker genes encode an altered target that is not sensitive to the inhibitor. An example of a gene for a selection marker for plant transformation is the BAR or PAT gene, which is used with the bialaphos selection agent. Spencer et al., J. Theor. Appl'd Genetics 79: 625-631 (1990). Another gene of a very useful selection marker is the neomycin phosphotransferase II (nptll) gene, isolated from Tn5, which confers resistance to kanamycin when it is under the control of plant regulatory signals. Fraley eí al., Proc. Nat'l Acad. Sci. (USA) 80: 4803 (1983). The hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin, is another example of a selectable marker that is useful. Vanden Elzen et al., Plant Mol. Biol. 5: 299 (1986). Other genes of positive selectable markers of bacterial origin that confer resistance to antibiotics include gentamicin acetyltransferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyltransferase and the determinant of bleomycin resistance. Hayford et al., Plant Physiol. 86: 1216 (1988); Jones went to., Mol. Gen. Genet. 210: 86 (1987); Svab eí al., Plant Mol. Biol. 14: 197 (1990); Hille eí al., Plant Mol. Biol. 7: 171 (1986). Other genes of positive selectable markers for plant transformation are not of bacterial origin. These genes include mouse dihydrofolate reductase, plant 5-enolpiruvilshiquimato-3-phosphate synthetase and plant acetolactate synthetase. Eichholtz et al., Somatic Cell Mol. Genet 13: 67 (1987); Shah e al., Science 233: 478 (1986); Charest i went to., Plant Cell Rep. 8: 643 (1990). Another class of marker genes useful for the transformation of plants with the DNA sequence requires the search and examination of presumably transformed plant cells in place of a direct genetic selection of the transformed cells for resistance to a toxic substance, such as an antibiotic These genes are particularly useful for quantifying or visualizing the spatial expression pattern of the DNA sequence in specific tissues and are often referred to as reporter genes because they can be fused to a gene or regulatory genetic sequence to investigate gene expression. Genes commonly used to examine presumably transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, Plant Mol. Biol. Rep. 5: 387 (1987); Teeri ei al., EMBO J. 8: 343 (1989); Koncz eí al., Proc. Nati Acad. Sci. (USA) 84: 131 (1987); From Block et al., EMBO J. 3: 1681 (1984). Another approach to identify relatively rare transformation events has been the use of a gene encoding a dominant constitutive regulator of the anthocyanin pigment pathway of Zea mays (Ludwig et al., Science 247: 449 (1990)). The appropriate DNA sequence can be inserted into the vector using any of several well-known and routine techniques. In general, the DNA sequence to be expressed will be linked to an expression vector by pooling said DNA sequence and said expression vector with one or more restriction endonucleases and then joining the restriction fragments together using the T4 DNA ligase. The sequence can be inserted with a direct or inverse orientation. The restriction and ligation procedures that can be used for tpurpose they are well known and routine for specialists. Suitable methods for t and for constructing expression vectors using alternative techniques, which are also well known and routine for the specialists, are described in great detail in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The polynucleotide of the invention, which encodes the heterologous structural sequence of a polypeptide of the invention, will generally be inserted into the vector using standard techniques so that it will be operably linked to the promoter for expression. The polynucleotide will be located such that the transcription start site will be located approximately 5 'with respect to a ribosome binding site. Said ribosome binding site will be located 5 'to the codon AUG that initiates the translation of the polypeptide to be expressed. In general, there will be no other open reading frame that begins with a start codon, usually AUG, and will be located between the ribosome binding site and said start codon. In addition, in general, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal in the constructs to be used in eukaryotic hosts. Transcription termination signals properly disposed by the 3 'end of the transcribed region in the construction of the polynucleotide will also be included. The vector containing the appropriate DNA sequence described elsewhere herein, as well as an appropriate promoter and other suitable control sequences, can be introduced into the appropriate host using various well-known techniques that are suitable for expressing in said host the desired polypeptide. The present invention also relates to host cells containing the constructions described above. The host cell can be a higher eukaryotic cell, such as a plant cell or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construction into the host cell can be carried out by calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transfection mediated by cationic lipids, electroporation, transduction, charge by scraping, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Representative examples of suitable hosts include bacterial cells, such as Streptococcus, Staphylococcus, E. coli, Streptomyces and Salmonella typhimurium cells.; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as cells of Drosophila S2 and Spodoptera Sf9; cells from other animals, such as CHO, COS and Bowes melanoma cells; and plant cells. Plant cells can derive from a wide range of plant types, in particular from monocotyledons, such as Graminiae family species including Sorghum bicolor and Zea mays, as well as dicotyledonous, such as soybean (Glycine max) and cañola (Brassica napus) , Brassica rapa ssp.). Preferably, the plants include corn, soybean, sunflower, safflower, barley, wheat, barley, rye, alfalfa and sorghum; however, the isolated nucleic acid and the proteins of the present invention can be used in species of the genera: Ananas, Antirrhinum, Arabidopsis, Arachis, Asparagus, Atropa, Oats, Brassica, Bromus, Browaalia, Camellia, Capsicum, Ciahorium, Citrus , Cocos, Cofea, Cucumis, Cucurbita, Datura, Daucus, Digitalis, Ficus, Fragaria, Geranium, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomoea, Juglans, Lactuca, Linum, Lolium, Lotus, Lycopersicon, Majorana, Mangifera , Manihot, Medicago, Musa, Nemesis, Nicotiana, Olea, Onobrychis, Oryza, Panieum, Pelargonium, Pennisetum, Persea, Petunia, Phaseolus, Pisum, Psidium, Ranunculus, Raphanus, Rose, Salpiglossis, Sécale, Senecio, Solanum, Sinapis, Sorghum , Theobroma, Triticum, Trifolium, Trigonella, Vigna, Vitis and Zea. The promoter regions of the invention can be isolated from any plant, including, for example, maize (Zea mays), cañola (Brassica napus, Brassica rapa ssp.), Alfalfa (Medicago sativa), rice (Oryza sativa), rye (Sécale). cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton ( Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), Coco (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), Cacao (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), oats, barley, vegetables, ornamentals and conifers Preferably, the plants include corn, soybean, sunflower, safflower, cañola, wheat, barley, rye, alfalfa and sorghum. Guests of a wide variety of expression constructs are well known and specialists will be able, with the present disclosure, to select a host to express the polypeptide according to this aspect of the present invention. The manipulated host cells can be cultured in conventional nutrient media, which can be modified as needed to, among others, activate promoters, select transformants or amplify genes. The culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression will generally be suitable for expressing the polypeptides of the present invention as will be apparent to those skilled in the art. The mature protein can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce said proteins using RNA derived from the DNA constructs of the present invention. After transformation of a suitable host strain and growth of said host strain to an appropriate cell density, when the selected promoter is inducible, it will be induced using the appropriate medium (e.g., temperature change or exposure to a chemical inducer) and The cells will be grown for an additional period. The cells are then harvested, typically by centrifugation, broken with physical or chemical means, and the resulting crude extract is retained for further purification. The microbial cells used in the expression of proteins can be broken using any convenient method, including freeze-thaw cycles, sonication, mechanical disruption or the use of cell lysate agents; said methods are well known to those skilled in the art. Plant transformation methods: The isolated nucleic acids of the present invention can be introduced into plants according to techniques known in the art. In general, the recombinant expression cassettes previously described and suitable for transforming plant cells are prepared. The techniques for transforming a wide variety of higher plant species are well known and are described in the technical literature, scientific and patent. See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). For example, the DNA construct can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, distribution with silicone fibers or microinjection of plant cell protoplasts or embryogenic calli. Alternatively, the DNA constructs can be combined with suitable DNA-T flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the host Agrobacterium tumefaciens will direct the insertion of the construct and the adjacent marker into the DNA of the plant cell when the cell is infected by the bacteria. See, U.S. Pat. N °: 5,691,616. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3: 2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Nati Acad. Sci (USA) 82: 5824 (1985). Ballistic transformation techniques are described in Klein et al., Nature 327: 70-73 (1987) and by Tomes, D. et al., In: Plant Cell, Tissue and Organ Culture: Fundamental Methods, Eds. O.L. Gamborg and G.C. Phillips, Chapter 8, pgs. 197-213 (1995). (See also Tomes et al., U.S. Patent Nos .: 5,886,244, 6,258,999, 6,570,067, 5,879,918) Transformation techniques mediated by Agrobacterium tumefaciens are well described in the scientific literature. See, for example, Horsch et al., Science 233: 496-498 (1984), and Fraley et al., Proc. Nati Acad. Sci (USA) 80: 4803 (1983). Although Agrobacterium is primarily useful in dicots, it is possible to transform some monocotyledons with Agrobacterium. For example, transformation with corn Agrobacterium is described in U.S. Pat. No.: No. 5,550,318. Other methods of transfection or transformation include (1) transformation mediated by Agrobacterium rhizogenes (see, for example, Lichtenstein and Fuller En: Genetic Engineering, vol 6, PWJ Rigby, Ed., London, Academic Press, 1987, and Lichtenstein, OR P., and Draper, J, in: DNA Cloning, Vol. II, DM Glover, Ed., Oxford, IRI Press, 1985), Application PCT / US87 / 02512 (WO 88/02406, published on April 7, 1988) describe the use of strain A4 of A. rhizogenes and its Ri plasmid together with vectors pARC8 or pARC16 of A. tumefaciens (2) liposome-mediated DNA uptake (see, eg, Freeman et al., Plant Cell Physiol., 25: 1363, 1984), (3) the vortex method (see, for example, Kindle, Proc. Nati Acad. Sci. (USA) 87: 1228, (1990). enter into plants by transfer direct DNA in pollen as described by Zhou et al., Methods in Enzymology, 101: 433 (1983); D. Hess, Intern. Rev. Cytoi, 107: 367 (1987); Luo ei al., Plant Mol. Biol. Repórter, 6: 165 (1988). The expression of the genes encoding the polypeptides can be achieved by injecting the DNA into the reproductive organs of a plant as described by Pena et al., Nature 325: 274 (1987). It is also possible to introduce the DNA by direct injection into the cells of immature embryos and subsequent rehydration of the dissected embryos as described by Neuhaus et al., Theor. Appl. Genet., 75: 30 (1987); and Benbrook went to., in Proceedings Bio Expo. 1986, Butterworth, Stoneham, Mass., Pgs. 27-54 (1986). A large variety of plant viruses that can be used as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, bromine mosaic virus and tobacco mosaic virus. Regeneration of the transformed plants The transformed plant cells obtained by any of the aforementioned transformation techniques can be cultured to regenerate an entire plant possessing the transformed genotype. Said regeneration techniques are often based on the manipulation of certain phytohormones in the growth medium of the tissue culture, typically based on a biocidal marker and / or herbicide that has been introduced together with a polynucleotide of the present invention. For the transformation and regeneration of corn see, for example, U.S. Pat. No. 5,736,369.

Plant cells transformed with a plant expression vector can be regenerated, for example, from individual cells, callus tissue or leaf discs using plant tissue culture techniques standard. It is well known in the art that it is possible to successfully grow cells, tissues and organs from practically any plant in order to regenerate the entire plant. The regeneration of plants from cultured protoplasts is described in Evans et al., Protoplast Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pgs. 124-176 (1983); and Union, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pgs. 21-73 (1985). Regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., Science, 227: 1229-1231 (1985). In this process, the transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species that is being transformed as described by Fraley et al., Proc. Nati Acad. Sci. (USA)., 80: 4803 (1983). This procedure typically produces shoots within two to four weeks and these shoots of the transformants are then transferred to an appropriate root inducing medium containing the selection agent and an antibiotic to prevent bacterial growth. The transgenic plants of the present invention can be fertile or sterile. Regeneration can also be achieved from calluses, explants or plant organs or parts thereof. Such regeneration techniques are generally described in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants either from individual plant protoplasts or from numerous explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This process of regeneration and growth includes the steps of selection of the cells and shoots of the transformant, rooting of said shoots of the transformant and growth of the seedlings on land. For the cultivation and regeneration of maize cells see in general, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988). The specialist will understand that once the cassette of recombinant expression has been incorporated in stable form in the transgenic plants and that its functionality has been confirmed, it is possible to introduce it in other plants by sexual crossing. Any of the numerous standard breeding techniques can be used, depending on the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by taking grafts or by tissue culture techniques to produce multiple identical plants. A selection of the desired transgenics is made and new varieties are obtained that will propagate vegetatively for commercial purposes. In seed-propagated crops, mature transgenic plants can self-cross to produce a homozygous inbred plant. The inbred plant produces seeds that contain the newly introduced heterologous nucleic acid. These seeds can be grown to obtain plants that will produce the selected phenotype. Mature transgenic plants can also be crossed with other appropriate plants, generally another inbred or hybrid, including, for example, an untransformed isogenic endogenous.

The parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruits and the like are also included in the invention, provided that these parts comprise cells containing the isolated nucleic acid of the present invention. Progeny and variants and mutants of regenerated plants are also included within the scope of the invention, provided that these plants comprise the introduced nucleic acid sequences. Transgenic plants expressing the selectable marker can be screened to verify the transmission of the nucleic acid of the present invention, for example, by immunoblotting and standard DNA detection techniques. The transgenic lines are also typically evaluated according to the expression levels of the heterologous nucleic acid. The expression at the RNA level can be determined initially to identify and quantify positive expression plants. Standard techniques for analyzing RNA can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA tempers and solution hybridization assays using probes specific for the heterologous nucleic acid. RNA positive plants can then be analyzed for protein expression by western blot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunohistochemistry can be used according to standard protocols using polynucleotide probes and heterologous nucleic acid specific antibodies, respectively, to localize the expression sites within the transgenic tissue. In general, numerous transgenic lines are examined by the presence of the incorporated nucleic acid in order to identify and select the plants with the most appropriate expression profiles. Some embodiments comprise a transgenic plant that is homozygous for the introduced heterologous nucleic acid; that is, a transgenic plant that contains two aggregated nucleic acid sequences, a gene at the same locus on each chromosome of a pair of chromosomes. The homozygous transgenic plant can be obtained by sexual crossing (autocross) of a heterozygous transgenic plant (or hemizygous) containing a single heterologous nucleic acid added, germination of some of the seeds produced and analysis in the resulting plants produced of an altered expression of the polynucleotides of the present invention in relation to a control (i.e., native, non-transgenic) plant. Backcrossing with a parent plant and crossing with a non-transgenic plant or with a transgenic plant for the same characteristic or for other characteristics is also contemplated. It is also considered that the transformed plants may be used in traditional breeding programs, including the TOPCROSS pollination systems described in US Pat. Nos .: 5,706,603 and 5,704,160, the contents of which are incorporated herein by reference. Assays with polynucleotides This invention also relates to the use of polynucleotides of cytokinin biosynthesis enzymes in markers to assist in breeding programs, as described, for example, in PCT Publication US89 / 00709. The DNA can be used directly in a detection or can be amplified enzymatically using PCR (Saiki et al., Nature 324: 163-166 (1986)) before the analysis. The RNA or cDNA can also be used in the same way. By way of example, PCR primers complementary to the nucleic acid encoding the enzymes of cytokinin biosynthesis can be used to identify and analyze the presence and expression of enzymes of cytokinin biosynthesis. With PCR, the gene present in a particular tissue or plant variety can be characterized by an analysis of the genotype of said tissue or variety. For example, deletions and insertions can be detected by a change in the size of the amplified product compared to the genotype of a reference sequence. Point mutations can be identified by hybridization of the amplified DNA with the RNA of radiolabeled cytokinin biosynthesis enzymes or, alternatively, with antisense DNA sequences of radiolabeled cytokinin biosynthesis enzymes. It is possible to distinguish perfectly matching sequences of the mismatched pairs by digestion with RNase A or by the differences in the melting temperatures. Sequence differences between a reference gene and genes that contain mutations can also be revealed by direct DNA sequencing. In addition, cloned segments of DNA can be used as probes to detect specific DNA segments. It is possible to improve the sensitivity of such methods with the appropriate use of PCR or another method of amplification. For example, a sequencing primer is used with the double-stranded product of the PCR or a single-stranded template molecule generated with a modified PCR. The determination of the sequence is carried out using conventional procedures with radioactively labeled nucleotides or by automatic sequencing procedures with fluorescent labels.

The determination of the genotype of the various plant varieties based on differences in the DNA sequence can be carried out detecting alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Deletions and insertions of small sequences can be visualized by high resolution gel electrophoresis. DNA fragments from different sequences can be distinguished in denaturing formamide gradient gels in which the mobilities of different DNA fragments in the gel are retarded in different positions according to their specific or partial melting temperatures (see, for example , Myers et al., Science, 230: 1242 (1985)). Sequence changes at specific locations can also be revealed in nuclease protection assays, such as RNase and S1 protection or with the chemical cleavage method (eg, Cotton et al., Proc. Nati Acad. Sci., ( USA), 85: 4397-4401 (1985)). Therefore, it is possible to detect a specific DNA sequence using methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or by the use of restriction enzymes, (for example, with restriction fragment length polymorphisms). ("RFLP")) and Southern blot of genomic DNA. In addition to gel electrophoresis and sequencing techniques More conventional DNA, mutations can also be detected by in situ analysis. It is possible to determine a mutation, for example, with a DNA sequencing assay. The samples are processed using methods known in the art to capture the RNA. The first cDNA chain is synthesized from of RNA samples by adding an oligonucleotide primer consisting of sequences that hybridize to a region of the mRNA. Reverse transcriptase and deoxynucleotides are added to allow synthesis of the first strand of the cDNA. The primer sequences are synthesized on the basis of the DNA sequences of the cytokinin modulating enzymes of the invention. The primer sequence generally comprises at least 15 consecutive bases and may contain at least 30 or even 50 consecutive bases. Cells that contain mutations or polymorphisms in the gene of the present invention can also be detected at the DNA level using various techniques. The DNA can be used directly in the detection or can be amplified enzymatically using PCR (Saiki et al., Nature, 324: 163-166 (1986)) before analysis. RT-PCR can also be used to detect mutations. It is particularly preferred to use RT-PCR in conjunction with automatic detection systems, such as, for example, GeneScan. The RNA or cDNA can also be used for the same purpose, with PCR or RT-PCR. By way of example, PCR primers complementary to the nucleic acid encoding the enzymes of cytokinin biosynthesis can be used to identify and analyze the mutations. Examples of representative primers are shown below. For example, deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridization of the amplified DNA with radioactively labeled RNA or, alternatively, with radioactively labeled antisense DNA sequences. Although perfectly matching sequences can be distinguished from unmatched pairs by digestion with RNase A or differences in temperatures of fusion, point mutations are preferably identified by sequence analysis. The primers used for the detection of mutations or polymorphisms in the ipt gene are: 5'GCGTCCAATGCTGTCCTCAACTA 3'5'GCTCTCCTCGTCTGCTAACTCGT3 'The above primers can be used to amplify the cDNA of the cytokinin biosynthesis enzymes or isolated genomic clones of a sample derived from an individual plant. The invention also provides the above primers in which 1, 2, 3 or 4 nucleotides of the 5 'and / or 3' end have been deleted. The primers can be used to amplify the gene isolated from the individual plant in such a way that it can then be subjected to various techniques to elucidate the DNA sequence. In this way it is possible to identify mutations in the DNA sequence. Assays with polypeptides The present invention also relates to diagnostic assays, such as quantitative and diagnostic assays for detecting the levels of cytokinin biosynthesis enzymes in cells and tissues, including the determination of normal and abnormal levels. Therefore, for example, a diagnostic assay according to the invention can be used to detect the expression of cytokinin biosynthesis enzymes compared to normal tissue samples to detect unacceptable levels of expression. The assay techniques that can be used to determine the levels of polypeptides of the present invention in a sample derived from a plant source are well known to those skilled in the art. Sayings Methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Among them, ELISAs are often preferred. An ELISA assay comprises initially the preparation of an antibody specific for the polypeptide, preferably a monoclonal antibody. In addition, an informant antibody that will bind to the monoclonal antibody is generally prepared. The reporter antibody binds to a detectable reagent, such as a radioactive, fluorescent or enzymatic reagent, in this example the horseradish peroxidase enzyme. To carry out the ELISA, a sample is removed from the host and incubated on a solid support, for example, a polystyrene disk, which binds to the proteins in the sample. Then all the free protein binding sites on the disk are covered by incubation with a non-specific protein, such as bovine serum albumin. Next, the monoclonal antibody is incubated in the disk, during which time the monoclonal antibody binds to any of the enzymes of the cytokinin biosynthesis bound to the polystyrene disk. Unbound monoclonal antibodies are removed by washing with buffer solution. The reporter antibody bound to the horseradish peroxidase is placed on the disk, resulting in the binding of the informant antibody to all the monoclonal antibodies bound to the cytokinin biosynthesis enzymes. Reporting antibodies that did not bind are removed by washing. Then the reagents are added to determine the peroxidase activity, including a colorimetric substrate, to the disc. The immobilized peroxidase linked to the enzymes of cytokinin biosynthesis through the primary and secondary antibodies produces a colored reaction product. The amount of color developed in a given period of time indicates the amount of enzymes of cytokinin biosynthesis present in the sample. Quantitative results are typically obtained with reference to a standard curve. A competition assay can be employed when antibodies specific for the cytokinin biosynthesis enzymes are attached to a solid support and the labeled enzyme derived from the host is passed through said solid support. The amount of detected label that is bound to the solid support can be correlated with the amount of enzymes of cytokinin biosynthesis present in the sample. Antibodies The polypeptides, its fragments or other derivatives or analogs thereof or the cells expressing them can be used as immunogens to produce antibodies against them. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments or the product of an Fab expression library. Several of the methods known in the art can be used for the production of said antibodies and fragments. The antibodies generated against the polypeptides corresponding to the sequence of the present invention can be obtained by direct injection of the polypeptides in an animal or by administration of the polypeptides to an animal, preferably a non-human animal. The antibody obtained in this manner will then bind to the polypeptides themselves. In this way, it is still possible to use a sequence that only encodes a fragment of the polypeptide to generate antibodies that bind to the entire native polypeptide. These antibodies are they can then be used to isolate the polypeptide from the tissue expressing said polypeptide. For the preparation of the monoclonal antibodies, any technique allowing to obtain antibodies produced by continuous culture of cell lines can be used. Examples include the hybridoma technique (Kohier, G. and Milstein, C, Nature 256: 495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4 : 72 (1983)) and the EBV hybridoma technique to produce human monoclonal antibodies (Colé et al., Pp. 77-96 in MONOCLONAL ANTIBODY AND CANCER THERAPY, Alan R. Liss, Inc. (1985)). Hybridoma cell lines secreting monoclonal antibodies constitute another aspect of this invention. The techniques described for the production of single chain antibodies (US Patent No. 4,946,778) can be adapted to produce single chain antibodies against the immunogenic polypeptide products of this invention. In addition, transgenic mice or other organisms, such as other mammals, can be used to express humanized antibodies against the immunogenic polypeptide products of this invention. The antibodies described above can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide of the present invention by binding said antibody to a solid support for isolation and / or purification by affinity chromatography. The polypeptide derivatives include antigenic or immunologically equivalent derivatives that make up a particular aspect of this invention.

The term "antigenically equivalent derivative", as used herein, encompasses a polypeptide, or its equivalent, which will be specifically recognized by certain antibodies which, when generated against the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between the antibody and its known antigen. The term "immunologically equivalent derivative", as used herein, encompasses a peptide, or its equivalent, which, when used in a suitable formulation to generate antibodies in a vertebrate, results in antibodies that act interfering with the interaction immediate physics between the antibody and its known antigen. The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof, is used as an antigen to immunize a mouse or other animal, such as a rat, guinea pig, goat, rabbit, sheep, bovine or chickens The fusion protein can provide stability to the polypeptide. The antigen can be associated, for example by conjugation, with an immunogenic carrier protein, for example bovine serum albumin (BSA) or limpet hemocyanin (KLH). Alternatively, a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof, may be sufficiently antigenic to enhance immunogenicity in order to obviate the use of a vehicle. Alternatively, phage display technology could be used to select for antibody genes with binding activities against the polypeptide from either the gene repertoires and amplified by PCR from human lymphocytes possessing anti-Fbp or libraries that were never exposed (McCafferty, J. ef al., (1990), Nature 348: 552-554; Marks, J. ef al., (1992) Biotechnology 10: 779-783). The affinity of these antibodies can also be increased by chain shuffling (Clackson, T. eí al., (1991) Nature 352: 624-628). The antibody must be re-evaluated for its high affinity for the polypeptide and / or the fusion protein. As mentioned above, a fragment of the final antibody can be prepared. The antibody can be either an intact antibody with a Mr <or about 150,000 or a derivative thereof, for example a Fab fragment or an Fv fragment as described in Sierra, A and Pluckthun, A., Science 240: 1038-1040 (1988). If there are two antigen binding domains, each domain can be directed against a different epitope, which is why they are called 'bispecific' antibodies. The antibody of the invention, as mentioned above, can be prepared by conventional means, for example with the established monoclonal antibody technology (Kohier, G. and Milstein, O, Nature, 256: 495-497 (1975)) or the use of recombinant media, for example. combinatorial libraries, for example as described in Huse, W.D. e. al., Science 246: 1275-1281 (1989). Preferably the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system, as previously described for the expression of the polypeptides of the invention. The choice of vector for the expression system will be determined partly according to the host, which may be a prokaryotic cell, such as E. coli (preferably of strain B) or Streptomyces sp. or a eukaryotic cell, such as mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell. The host may also be a transgenic animal or a transgenic plant for example as described in Hiatt, A. et al., Nature 340: 76-78 (1989). Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived, for example, from baculovirus and vaccinia. The Fab fragment can also be prepared from its parent monoclonal antibody by treatment with enzymes, for example using papain to cleave the Fab portion of the Fe moiety. Binding molecules and assays with the enzymes of cytokinin biosynthesis This invention also provides a method of identifying molecules, such as binding molecules, that bind to the enzymes of cytokinin biosynthesis. The genes encoding proteins that bind to enzymes, such as binding proteins, can be identified by numerous methods known to those skilled in the art, for example, ligand panning and FACS separation. Such methods are described in many of the laboratory manuals such as, for example, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991). For this purpose, for example, cloning of the expression can be used. For this purpose, polyadenylated RNA is prepared from a cell that expresses the enzymes of cytokinin biosynthesis, a cDNA library is generated from this RNA, the library is divided into clusters and the clusters are transfected individually into cells that they do not express the enzyme. The cells transfected are then exposed to the labeled enzyme. The enzyme can be labeled using various well-known techniques, including standard methods of radioiodination or inclusion of a recognition site for a site-specific protein kinase. After exposure, the cells are fixed and the binding of the enzyme is determined. These procedures are conveniently carried out on glass slides. The cDNA pools produced by the cells that were linked to the enzymes of cytokinin biosynthesis are identified. Subgroupings are prepared from these positives, transfected into host cells and evaluated as previously described. The use of an iterative sub-aggregation and re-evaluation process allows the isolation of one or more individual clones that encode the putative binding molecule. Alternatively, a ligand labeled by photoaffinity can be ligated to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule with which it binds, such as a binding molecule. The material bound by cross-linking is resolved with electrophoresis on polyacrylamide gel ("PAGE") and then exposed to an X-ray film. The labeled complex containing ligand-binding can be trimmed, resolved into peptide fragments and then subjected to to protein microsequencing. The amino acid sequence obtained with the microsequencing can be used to design single or degenerate oligonucleotide probes to examine cDNA libraries in order to identify genes encoding the putative binding molecule. The polypeptides of the invention can also be used to evaluate the enzyme binding capacity of cytokinin biosynthesis of molecules of enzyme binding of cytokinin biosynthesis, such as binding molecules, in cells or in cell-free preparations. The polypeptides of the invention can also be used to evaluate the binding of substrates and ligands of small molecules in, for example, cells, cell-free preparations, chemical libraries and mixtures of natural products. These substrates and ligands can be natural substrates and ligands or can be structural or functional mimetics. Antibodies against cytokinin biosynthesis enzymes represent a useful class of binding molecules contemplated by this invention. Antagonists and agonists: assays and molecules The invention also provides a method for evaluating compounds to identify those that enhance or block the action of cytokinin biosynthesis enzymes on cells., such as interaction with substrate molecules. An antagonist is a compound that decreases the natural biological functions of enzymes. A particular enzyme for this type of targeting is cytokinin oxidase. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind cytokinin oxidase and thereby inhibit or cancel their activity. Potential antagonists may also be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody, which binds to the same sites in the binding molecule, such as a cytokinin oxidase binding molecule, without inducing the activities induced by enzymes of cytokinin metabolism, which prevents the action of the enzyme by exclusion of the binding enzyme.

Potential antagonists include a small molecule that binds and occupies the binding site of the polypeptide thereby preventing binding to cell binding molecules, such as binding molecules, so that normal biological activity is prevented. Examples of small molecules include, for example, small organic molecules, peptides or peptide-like molecules. Other potential antagonists include molecules that affect the expression of the gene encoding enzymes of cytokinin biosynthesis (eg, transactivation inhibitors). Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through the formation of double or triple helices. Antisense techniques are described, for example, in: Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENETIC EXPRESSION, CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, in Lee et al., Nucleic Acids Research 6: .3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). The methods are based on the binding of a polynucleotide to a complementary DNA or RNA. For example, the 5 'coding portion of a polynucleotide encoding the mature polypeptide of the present invention can be used to design an antisense RNA oligonucleotide from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to the region of the gene involved in transcription, thereby preventing transcription and enzyme production of cytokinin biosynthesis. The antisense RNA oligonucleotide hybridizes with the mRNA in vivo and blocks the translation of the mRNA molecule into the enzymes of the biosynthesis of cytokinins. The previously described oligonucleotides can also be distributed in the cells, so that said antisense RNA or DNA can be expressed in vivo to inhibit the production of cytokinin biosynthesis enzymes. The DNAs of this invention can also be used to cosuppress or silence the genes of the cytokinin metabolism enzymes; for example, as described in the publication of Patent Application WO 98/36083. Antagonists can be used for example to increase the levels of cytokinins and / or decrease the auxins available in plant cells. As an alternative, this invention provides methods for evaluating agonists, which are those molecules that act by increasing the natural biological function of the enzymes. Targets of this type include enzymes such as ipt, β-glucosidase and iaa-1. Potential agonists include small organic molecules, peptides, polypeptides and antibodies that bind to biosynthetic enzymes and thereby stimulate or increase their activity. Potential agonists may also be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds to sites on a binding molecule, such as an ipt binding molecule and promotes enzyme-induced activities of metabolism of cytokinins, thereby improving the action of the enzyme. Potential agonists include small molecules that bind to and occupy the allosteric sites of the enzyme thereby promoting binding to cellular binding molecules, such as substrates, thereby increasing the normal biological activity. Examples of small molecules include, for example, small organic molecules, peptides or peptide-like molecules. Other potential agonists include molecules that affect the expression of the gene encoding the enzymes of cytokinin biosynthesis (eg transactivators). "Stacking" of constructs and features In certain embodiments the nucleic acid sequences of the present invention can be used in combination ("stacked") with other polynucleotide sequences of interest in order to create plants with a desired phenotype. The polynucleotides of the present invention can be stacked with any gene or combination of genes, and the combinations generated can include multiple copies of one or more of the polynucleotides of interest. The desired combination can affect one or more characteristics; that is, certain combinations can be created to modulate gene expression that affects the activity of cytokinins. For example, sensitization of cytokinin synthesis can be combined with sensitization of cytokinin oxidase expression. Other combinations can be designed to produce plants with a variety of desired characteristics, including, for example, desirable characteristics for animal feeds, such as oil-rich genes (eg, US Patent No. 6,232,529). ); balanced amino acids (eg hordothionines (U.S. Patent Nos .: 5,990,389; 5,885,801; 5,885,802; and 5,703,409); lysine-rich barley (Williamson et al. (1987) Eur. J. Biochem 165: 99-106, and WO 98/20122), and methionine-rich proteins (Pedersen et al (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); greater digestibility (e.g., modified storage proteins (U.S. Patent Application Number: 10 / 053,410, filed November 7, 2001); and thioredoxin (U.S. Patent Application No. 10) / 005,429, filed December 3, 2001)), the contents of which are incorporated herein by reference. The polynucleotides of the present invention can also be stacked with desirable characteristics for resistance to insects, diseases or herbicides (eg, Bacillus thuringiensis toxic proteins (U.S. Patent Nos .: 5,366,892; 5,747,450; 5,737. 514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48: 109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825), fumonisin detoxification genes (Patent of U.S. Patent No. 5,792,931), avirulence resistance genes and diseases (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089), acetolactate synthetase (ALS) mutants leading to resistance to herbicides, such as S4 and / or Hra mutants, glutamine synthetase inhibitors such as phosphinothricin or basta (eg, the bar gene); resistance to glyphosate (EPSPS gene)); and desirable characteristics for processing products or processes such as high oil content (e.g., U.S. Patent No.: 6,232,529); modified oils (e.g., fatty acid desaturases genes (U.S. Patent No. 5,952,544; WO 94/11516)); modified starches (for example, ADPG pyrophosphorylases (AGPase), starch synthetase SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Patent No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthetase and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHA), the contents of which are incorporated herein by reference. Polynucleotides of the present invention could also be combined with polynucleotides that affect agronomic characteristics, such as male sterility (e.g., see U.S. Patent No. 5,583,210), stem length, timing of flowering or transformation technology characteristics such as cell cycle regulation or gene targeting (eg, WO 99/61619; WO 00/17364; WO 99/25821), the contents of which are incorporated herein by reference. These stacked combinations can be created by any method, including for example, crossing plants with any conventional methodology or TopCross or genetic transformation. If the characteristics were stacked by genetic transformation of the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more of the desired characteristics may be used as a target to introduce other characteristics through a subsequent transformation. The characteristics can be introduced simultaneously into a cotransformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences are introduced, the two sequences may be contained in separate transformation (trans) cassettes or may be contained in the same transformation cassette (cis). The expression of the sequences of interest can be directed by the same promoter or by different promoters.

In certain cases, it may be desirable to introduce a transformation cassette which will suppress the expression of a polynucleotide of interest. This can be accompanied by any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of characteristics in the plant. Use in breeding methods The transformed plants of the invention can be used in a plant breeding program. The goal of plant breeding is to combine, in a single variety or hybrid, various desirable characteristics. For field crops, these characteristics may include, for example, resistance to diseases and insects, tolerance to heat and drought, shorter time to maturity of crops, higher yield and better agronomic quality. With the mechanical harvesting of many crops, it is desirable to achieve a uniformity of the characteristics of the plants, such as germination and standing establishment, growth rate, maturity and height of plants and ears. Traditional plant breeding is an important tool for the development of new and improved cash crops. This invention encompasses methods for producing a maize plant by crossing a first progenitor maize plant with a second progenitor maize plant, where one or both parent maize plants is a transformed plant having greater vigor, as described in FIG. I presented. Plant breeding techniques known in the art and used in corn plant breeding programs include, for example, recurrent selection, volume selection, mass selection, backcross, breeding pedigree, breeding by open pollination, selection improved by length polymorphisms of restriction fragments, improved selection with genetic markers, duplicated haploids and transformation. Often combinations of these techniques are used. The development of corn hybrids in a corn plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines and the evaluation of the crosses. There are many analytical methods available to evaluate the outcome of a cross. The oldest and most traditional method of analysis is the observation of phenotypic characteristics. Alternatively, you can examine the genotype of a plant. It is possible to move a genetic characteristic that has been introduced by genetic engineering into a particular maize plant using transformation techniques, to another line using traditional breeding techniques that are well known in the art of plant breeding. For example, a backcrossing approach is commonly employed to move a transgene from a maize transformed plant to an elite inbred line and the resulting progeny could then comprise the transgene (s). In addition, if an inbred line was used for the transformation then the transgenic plants could be crossed with a different inbreeding in order to produce a hybrid transgenic maize plant. As used herein, the term "crossing" can refer to a simple X-by-Y cross, or to the backcrossing process, depending on the context. The development of a corn hybrid in a corn plant breeding program comprises three steps: (1) the selection of plants from various germplasm groupings for the first breeding crosses; (2) the cross-breeding of the plants selected from breeding crosses for several generations to produce a series of inbred lines, which, although differing from one another, are purebred and very uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids. During the process of inbreeding in corn, the vigor of the lines decreases. The vigor is restored when two different inbred lines are crossed to produce the hybrid. An important consequence of the homozygosity and homogeneity of inbred lines is that the hybrid created by crossing a pair of defined inbreds will always be the same. Once the inbreds that will originate a superior hybrid are identified, it is possible to reproduce the hybrid seeds indefinitely as long as the homogeneity of the inbred progenitors is maintained.

The transgenic plants of the present invention can be used to produce a single-cross hybrid, a three-way hybrid or a double cross hybrid. A single-cross hybrid is obtained when two inbred lines are crossed to produce the F1 progeny. A double-cross hybrid is produced from four inbred lines crossed in pairs (A x B and C x D) and then the two F1 hybrids (A x B) x (C x D) are crossed again. A hybrid of three-way crossing occurs from three inbred lines, where two of the inbred lines are crossed (A x B) and then the resulting F1 hybrid is crossed with the third inbred (A x B) x O Much of the vigor and uniformity of the hybrid exhibited by F1 hybrids are lost in the next generation (F2). As a result, the seeds produced by the hybrids are generally consumed before they are planted.

According to the invention, nucleotide sequences are provided which allow transcription to start in seeds. The sequences of the invention comprise the regions of initiation of transcription associated with the formation of seed and seed tissues. Therefore, the compositions of the present invention comprise novel nucleotide sequences as regulatory sequences. A method of expressing an isolated nucleotide sequence in a plant is provided using the transcription initiation sequences described herein. The proper techniques were described by Maniatis, T., Fritsch, E.F. and Sambrook, J. in MOLECULAR CLONING, A Laboratory Manual (2nd edition, 1989, Cold Spring Harbor Laboratory). The method comprises transforming a plant cell with a transformation vector comprising an isolated nucleotide sequence operably linked to the promoter of the present invention and regenerating a plant stably transformed from the transformed plant cell. In this way, the promoter is useful for controlling the expression of endogenous as well as exogenous products in a manner preferably with seeds. Under the regulation of the start of transcription, the promoter region with preference for seeds will be the sequence of interest, which will provide a modification of the phenotype of the seed. Said modification includes the modulation of the production of an endogenous product, with respect to quantity, relative distribution or the like, or the production of an exogenous expression product to provide a new function or product in the seeds. The term "with preference for seeds" refers to a favored expression in seeds, including at least one between embryo, grain, pericarp, endosperma, núcelo, aleurona, pedicel and similar. A "regulatory element" refers to the sequences responsible for the preferred tissue expression and temporally preferred of the associated coding sequence, including promoters, terminators, enhancers, introns and the like. A "promoter" refers to a region of regulatory DNA that usually comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may further comprise other recognition sequences generally located 5 'to the TATA box, called 5' promoter elements, which affect the rate of transcription initiation. If it is considered that having identified the nucleotide sequences for the promoter region described herein, the isolation and identification of other regulatory elements in the 5 'untranslated region located 5' to the region belongs to the state of the art. particular promoter identified herein. Therefore, the promoter region described herein is generally defined as comprising 5 'regulatory elements, such as those responsible for a preferred tissue expression and temporally preferred coding sequence, enhancers and the like. In the same way, promoter elements that allow expression in the desired tissue, such as seeds, can be identified, isolated and used with other nuclear promoters to confirm expression with preference for seeds. The isolated promoter sequences of the present invention can be modified to provide a range of expression levels of the sequence of nucleotides isolated. It can be used less than the complete promoter region and still retain the ability to direct an expression with preference for seeds. However, it is considered that it is possible to decrease the levels of mRNA expression with the deletion of portions of the promoter sequence. Therefore, the promoter can be modified to be a weak or strong promoter. In general, a "weak promoter" refers to a promoter that directs the expression of a coding sequence at a low level. A "low level" refers to levels between approximately 1 / 10,000 transcripts and approximately 1 / 100,000 transcripts and approximately 1 / 500,000 transcripts. Conversely, a strong promoter directs the expression of a coding sequence at a high level or at levels between about 1/10 transcripts and about 1/100 transcripts and about 1/1000 transcripts. In general, at least about 20 nucleotides of an isolated promoter sequence will be used to direct the expression of a nucleotide sequence. It is considered that to increase the levels of transcription, enhancers can be used in combination with the promoter regions of the invention. Enhancers are nucleotide sequences that act by increasing the expression of a promoter region. Enhancers are known in the art and include the region of the SV40 enhancer, the 35S enhancer element and the like. The promoter of the present invention can be isolated from the 5 'untranslated region flanking the transcription start site of its respective coding sequence. Also, the terminator can be isolated from the 3 'untranslated region flanking the stop codon of its respective coding sequence.

The term "isolated" refers to a material, such as a nucleic acid or a protein, that is: (1) substantially or essentially free of the components that usually accompany or interact with said material as it is in its natural environment or (2) if the material is in its natural environment, said material has been altered by human intervention deliberately in a composition and / or has been located in the locus of a cell other than the native locus for said material. Methods of isolation of promoter regions are well known in the art. The sequence of the eepl promoter region is shown in SEQ ID N °: 7. The sequence of the promoter region eep2 is shown in SEQ ID N °: 18. The eepl promoter is shown in SEQ ID N °: 7 has 960 nucleotides in length. There is a putative CAAT motif that is 308 bp located 5 'with respect to the start of the translation and a putative TATA motive 139 bp located 5' with respect to the start of the translation. The promoter was isolated from sequences. ESTs found in maize tissue libraries of 4 and 6 DAP embryo sacs, as well as complete grains of 5 and 7 DAP. The eepl promoter can handle expression problems by providing expression in seed tissues during the early stages of seed development. The eep2 promoter shown in SEQ ID NO: 18 is 1027 nucleotides in length. The promoter was isolated from an EST sequence found in embryonic sac tissue libraries of 4 DAP maize (days after pollination) and is highly specific for the early expression of grains and endosperm, determined by distribution of EST in the libraries and by determining MPSS Lynx profiles.

The promoter regions of the invention can be isolated from any plant, including, for example, maize (Zea mays), cañola (Brassica napus, Brassica rapa ssp.), Alfalfa (Medicago sativa), rice (Oryza sativa), rye (Sécale). cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton ( Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), Coco (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), Cacao (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), oats, barley, vegetables, ornamentals and conifers Preferably, the plants include corn, soybeans, sunflower, safflower, barley, wheat, barley, rye, alfalfa and sorghum. Promoter sequences from other plants can be isolated according to well-known techniques based on their sequence homology with the promoter sequences described herein. In these techniques, all or a portion of the promoter sequence known as a probe that will selectively hybridize with the other sequences present in a population of cloned fragments of genomic DNA (ie, genomic libraries) of a chosen organism can be used. The methods that are already available in the art for the hybridization of nucleic acid sequences to obtain sequences corresponding to the promoter of the present invention can be used. The whole promoter sequence or portions of the same as a probe capable of hybridizing specifically with the corresponding promoter sequences. To achieve specific hybridization under various conditions, such probes include sequences that are unique and that are preferably at least about 10 nucleotides in length and more preferably at least about 20 nucleotides in length. Said probes can be used to amplify the corresponding promoter sequences of a chosen organism using the well known polymerase chain reaction (PCR) process. This technique can be used to isolate additional promoter sequences from the desired organism or as a diagnostic assay to determine the presence of the promoter sequence in an organism. Examples include evaluation by hybridization of plated DNA libraries (either as plaques or colonies, see, for example, Innis et al. (1990 J PCR Protocols, A Guide to Methods and Applications, eds., Academic Press). the sequences corresponding to the promoter sequence of the present invention and which hybridize with the promoter sequence described herein will be at least 50% homologous, 55% homologous, 60% homologous, 65%> homologous, 70% homologous, 75 % homologous, 80% homologous, 85% homologous, 90% homologous, 95% homologous and 98% homologous or more, of the sequence described The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides : (a) "reference sequence", (b) "comparison window", (c) "percentage of sequence identity" and (d) "substantial identity." (a) As used in this document, a "reference sequence" it is a defined sequence that is used as a basis for the comparison of sequences. A reference sequence may be a subset or the entirety of a specified sequence; for example, a segment of a promoter sequence of a full length or the complete sequence of the promoter sequence. (b) As used herein, a "comparison window" refers to a contiguous and specified segment of a polynucleotide sequence, where the polynucleotide sequence can be compared to a reference sequence and where the portion of the Polynucleotide sequence in the comparison window may comprise additions or deletions (ie, mismatches or gaps) when compared to the reference sequence (which does not include additions or deletions) for an optimal alignment of the two sequences. In general, the comparison window is at least 20 contiguous nucleotides in length and, optionally, may be 30, 40, 50, 100 or more in length of contiguous nucleotides. It will be understood by those skilled in the art that in order to avoid a great similarity to the reference sequence due to the inclusion of mismatches in the polynucleotide sequence, a mismatch penalty is typically introduced and subtracted from the number of matches . (c) As used herein, the "percent sequence identity" refers to the value determined by comparing two sequences aligned optimally in a comparison window, where the portion of a polynucleotide sequence in the window comparison may comprise additions or deletions (i.e., mismatches) as compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. (d) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence possessing at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described with the standard parameters. Methods of sequence alignment for a comparison are well known in the art. Gene comparisons can be determined by BLAST searches (Basic Local Alignment Search Tool; Altschul, SF, et al., (1993) J. Mol. Bioi 215: 403-410; see also www.ncbi.nlm.nih. gov / BLAST /) with the default parameters in order to identify the sequences contained in the "GENEMBL" BLAST database. The sequence can be analyzed by its identity with all the DNA sequences available to the public contained in the GENEMBL database using the BLASTN algorithm with the predetermined parameters. The identity with the sequence of the present invention means that a polynucleotide sequence has at least 65% sequence identity, more preferably at least 70% identity. sequence, more preferably at least 75% sequence identity, more preferably at least 80% identity, more preferably at least 85% sequence identity, more preferably at least 90% % sequence identity and even more preferably at least 95% sequence identity, where said percentage of sequence identity is based on the entire promoter region. For the purpose of defining the present invention, the GAP (Global Alignment Program) is used. GAP employs the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of mismatches. GAP takes into account all the alignments and possible positions of the mismatches and creates an alignment with the highest number of matching bases and the least number of mismatches. It allows the provision of a limit of creation of mismatches and a limit of the extent of mismatches, expressed in units of matching bases. GAP must get a profit with the limit of creation of mismatches in match amounts for each mismatch it inserts. If a limit of the extent of mismatches greater than zero is chosen, GAP must also provide a gain for each mismatch inserted, whose length is the number of mismatches, with respect to the extension limit of the lack of coincidences. The default values for the mismatch creation limit and the mismatch extension limit in the Wisconsin Package® Version 10 (Accelrys, Inc., San Diego, CA) for Protein sequences are 8 and 2, respectively. For nucleotide sequences, the default mismatch creation limit is 50, while the default mismatch limit is 3. The limits of creation of mismatches and extension of mismatches can be expressed as an integer selected from the group of integers from 0 to 200. So, for example, the values of the limits of creation of mismatches and extension of the mismatches can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. GAP represents a member of the family of best alignments. This family can have many members, but no other member has a better quality. GAP shows four qualification values for the alignments: Quality, Relation, Identity and Similarity. Quality is the maximized metric to align the sequences. The Relationship is the quality divided by the number of bases in the shortest segment. Percentage Identity is the percentage of the symbols that actually match. Percentual Similarity is the percentage of symbols that are similar. The symbols that are crossed with respect to mismatches are not taken into account. A similarity is scored when the value of the qualification matrix for a pair of symbols is greater than or equal to 0.50, such as the similarity threshold. The rating matrix used in Version 10 of the Wisconsin Genetics software package is BLOSUM62 (see, Henikoff &; Henikoff (1989) Proc. Nati Acad. Sci. USA 89: 10915). Sequence fragments with a high percentage of identity with the sequences of the present invention also refer to those fragments of a particular promoter sequence described herein that works by promoting the seed expression of an isolated nucleotide sequence operatively linked to it. These fragments will comprise at least about 20 contiguous nucleotides, preferably at least about 50 contiguous nucleotides, more preferably at least about 75 contiguous nucleotides, even more preferably at least about 100 contiguous nucleotides of the particular promoter nucleotide sequence that is described herein. The nucleotides of said fragments will usually comprise the TATA recognition sequence of the particular promoter sequence. Said fragments can be obtained using restriction enzymes to cleave the natural promoter sequences described herein; synthesizing a nucleotide sequence from a natural DNA sequence; or through the use of PCR technology. See in particular, Mullis et al. (1987) Methods Enzymol. 155: 335-350, and Eriich, ed. (1989) PCR Technology (Stockton Press, New York). Again, variants of these fragments, such as those obtained by site-directed mutagenesis, are comprised in the compositions of the present invention. Nucleotide sequences comprising at least about 20 contiguous nucleotides of the sequence shown in SEQ ID NO: 10 are also included. These sequences can be isolated by hybridization, PCR and the like. Said sequences comprise fragments capable of directing an expression with preference for seeds, fragments that are useful as probes to identify similar sequences, as well as the elements responsible for temporal or tissue specificity.

Biologically active variants of the promoter sequence are also comprised in the compositions of the present invention. A "regulatory variant" is a modified form of a promoter in which one or more bases have been modified, deleted or added. For example, a common way of removing part of a DNA sequence is to use an exonuclease in combination with DNA amplification to produce unidirectional nested deletions of double-stranded DNA clones. There is a commercial item set for this purpose that is sold under the trade name Exo-Size ™ (New England Biolabs, Beverly, Mass.). Briefly, this method comprises the incubation of exonuclease III with DNA to progressively remove the nucleotides in the 3 'to 5' direction in the 5 'overlays, cohesive ends or nicks in the DNA annealing. However, exonuclease III can not remove the nucleotides in the superpositions of 4 3 'bases. The timed digestions of a clone with this enzyme produce unidirectional nested deletions. An example of a regulatory sequence variant is a promoter formed by one or more deletions in a larger promoter. It is possible to suppress the 5 'portion of a promoter to the TATA box near the transcription initiation site without abolishing the promoter activity, as described by Zhu et al., The Plant Cell 7: 1681-89 (1995). Said variants should retain the promoter activity, in particular the ability to direct expression in seeds or seed tissues. Biologically active variants include, for example, the native regulatory sequences of the invention that possess one or more substitutions, deletions or insertions of nucleotides. The activity can be measured by Northern blot analysis, measurements of informant activity when using transcription and similar fusions. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), incorporated herein by reference. The nucleotide sequences for the seed-preferred promoter described in the present invention, as well as the variants and fragments thereof, are useful in the genetic manipulation of any plant when they are operatively linked with an isolated nucleotide sequence whose expression must be controlled to obtain the desired phenotypic response. The term "operably linked" refers to the fact that the transcription or translation of the isolated nucleotide sequence is under the effect of the regulatory sequence. In this manner, the nucleotide sequence of the promoter of the invention can be provided in an expression cassette together with an isolated nucleotide sequence for expression in the plant of interest, more particularly in the seeds of the plant. Said expression cassette is provided with a plurality of restriction sites to insert the nucleotide sequence under the transcription control of the promoter. The genes of interest expressed under the direction of the promoter of the invention can be used to vary the phenotype of the seeds. This can be achieved by increasing the expression of endogenous or exogenous products in the seeds. Alternatively, results can be obtained by providing a reduction in the expression of one or more endogenous products, in particular enzymes or cofactors, in the seeds. These modifications result in a change in the phenotype of the transformed seeds. It is considered that The promoter can be used with its native coding sequence to increase or decrease expression, resulting in a change in the phenotype of the transformed seeds. General categories of genes of interest for the purposes of the present invention include, for example, those genes that are involved in the information, such as the zinc fingers; those involved in communication, such as kinases, and those involved in maintenance, such as heat shock proteins. The more specific categories of transgenes include, for example, genes that code for important traits for agronomy, insect resistance, disease resistance, herbicide resistance, and grain characteristics. Still other categories of transgenes include genes for inducing the expression of exogenous products, such as enzymes, cofactors and hormones from plants and other eukaryotic organisms as well as prokaryotes. It is considered that it is possible to operatively bind any gene of interest, including the native coding sequence, with the regulatory elements of the invention and express it in the seeds. Modifications that affect the characteristics of the grains include an increased content of oleic acid, altered levels of saturated or unsaturated fatty acids. Likewise, it may be convenient to increase the levels of amino acids containing lysine and sulfur, as well as a modification of the type and content of starch in the seeds. Modifications of the hordothionine protein are described in WO 9416078, filed April 10, 1997; WO 9638562, filed March 26, 1997; WO 9638563, filed March 26, 1997 and U.S. Pat. No.: 5,703,409, filed December 30, 1997; whose contents are incorporated in this document as a reference. Another example is the lysine and / or sulfur-rich protein of the seeds encoded by soy 2S albumin which is described in WO 9735023, filed March 20, 1996, and the barium chymotrypsin inhibitor, Williamson et al., ( 1987) Eur. J. Biochem. 765: 99-106, whose contents are incorporated in this document as a reference. Derivatives of the following genes can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley lysine-rich polypeptide (BHL) is derived from the barium chymotrypsin inhibitor, WO 9820133, filed November 1, 1996, the contents of which are incorporated herein by reference. Other proteins include methionine-rich plant proteins, such as sunflower seeds (Lilley et al., (1989), Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, Applewhite, H. (ed.); American Oil Chemists Society, Champaign, Illinois, 497-502, incorporated herein by reference)); of corn (Pedersen et al. (1986), J Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359, the contents of which are incorporated herein by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12: 123, incorporated herein by reference). The agronomic characteristics in seeds can be improved by altering the expression of the genes that: affect the growth response and development of the seeds during environmental stress, Cheikh-N eí al (1994) Plant Physiol. 106 (1): 45-51) and of the genes that control the metabolism of carbohydrates to reduce grain abortion in corn, Zinselmeier et al. (1995) Plant Physiol. 107 (2): 385-391. These genes include, for example, those encoding enzymes of cytokinin biosynthesis, such as isopentenyl transferase; genes encoding cytokinin catabolic enzymes, such as cytokinin oxidase; genes that encode the polypeptides involved in cell cycle regulation, such as Cyclin D or cdc25; genes encoding cytokinin receptors or sensors, such as CRE1, CKI1 and CKI2, histidine phosphotransmitters or regulators of cytokinin response. Insect resistance genes can encode resistance to pests that represent a large loss in yield, such as rootworms, cutworms, European corn borer and the like. Such genes include, for example, Bacillus thuringiensis endotoxin genes, US Pat. No. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109; lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825); and similar. Genes encoding disease resistance characteristics may include: detoxification genes, such as against fumonisin (WO 9606175, filed June 7, 1995); avirulence genes (avr) and disease resistance genes (R) (Jones et al., 1994, Science 266: 789; Martin et al., Science 262: 1432; Mindrinos et al., 1994, Cell 78: 1089); and similar. The commercial characteristics may also be encoded in one or several genes that could alter or increase, for example, starch for the production of paper, textiles and ethanol or provide the expression of proteins with other commercial uses. Another important commercial use of Transformed plants is the production of polymers and bioplastics as described in U.S. Pat. No. 5,602,321, filed February 11, 1997. Genes such as β-ketothiolase, PHBase (polyhydroxybutyrate synthetase) and acetoacetyl-CoA reductase (see Schubert et al., 1988, J Bacteriol 170: 5837-5847) they facilitate the expression of polyhydroxyalkanoates (PHA). Exogenous products include enzymes and plant products as well as those from other sources, including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones and the like. The level of proteins can be increased, in particular modified proteins that have a better distribution of amino acids to improve the nutritional value of the seeds. This is achieved with the expression of proteins that have a higher content of amino acids. The nucleotide sequence operably linked to the regulatory elements described herein may be an antisense sequence for the desired gene. The term "antisense DNA nucleotide sequence" refers to a sequence with reverse orientation with respect to the normal 5 'to 3' orientation of said nucleotide sequence. When distributed in the plant cell, the expression of the antisense DNA sequence prevents normal expression of the DNA nucleotide sequence for the desired gene. The antisense nucleotide sequence encodes an RNA transcript that is complementary and capable of hybridizing to the endogenous messenger RNA (mRNA) produced by transcription of the DNA nucleotide sequence of the desired gene. In this case, the production of the native protein encoded by the desired gene is inhibited to achieve the desired phenotypic response. Therefore, the promoter sequences described in this The document can be operably linked to antisense DNA sequences to reduce or inhibit the expression of a native protein in the seeds of the plant. The expression cassette will also include, at the 3 'terminal end of the isolated nucleotide sequence of interest, a transcription termination and functional translation region in plants. The termination region may be native with respect to the promoter nucleotide sequence of the present invention, it may be native with respect to the DNA sequence of interest, or it may be derived from another source. Other convenient termination regions can be obtained from the Ti plasmid of A. tumefaciens, such as the termination regions of octopine synthetase and nopaline synthetase. See also: Guerineau eí al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon eí al. (1991) Genes Dev. 5: 141-149; Mogen eí al. (1990) Plant Cell 2: 1261-1272; Munroe e to al. (1990) Gene 91: .151-158; You dance to the. 1989) Nucleic Acids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

The expression cassettes may also contain 5 'direct sequences. These guidelines can act to improve translation. Translation guidelines are known in the art and include: picornavirus guidelines, e.g., EMCV guideline (5 'non-coding region of encephalomyocarditis) Elroy-Stein et al., (1989) PNAS USA. 86: 6126-6130; Potivirus guidelines, for example, TEV (Tobacco Virus etch) guideline Allison et al., (1986); guideline MDMV (Virus in dwarf corn mosaic); Virology 154: 9-20), and heavy chain binding protein of human immunoglobulin (BiP), Macejak et al. (1991) Nature 353: 90-94; untranslated guideline of the envelope protein mRNA of the alfalfa mosaic virus (AMV RNA 4), Jobling et al, (1987) Nature 325: 622-625; Guideline of Tobacco Mosaic Virus (TMV), Gallie et al., (1989) Molecular Biology of RNA, pages 237-256; and guideline of corn chlorotic spotted virus (MCMV), Lommel et al. (1991) Virology 81: 382-385. See also, Della-Cioppa et al., (1987) Plant Physiology 84: 965-968. The cassette may also contain sequences that improve the translation and / or stability of the mRNA, such as introns. In those cases where it is desirable to direct the expressed product of the isolated nucleotide sequence to a particular organelle, in particular plastids, amyloplasts, or to the endoplasmic reticulum or to secrete it on the cell surface or extracellularly, the expression cassette may further comprise a sequence of coding a transit peptide. Such transit peptides are well known in the art and include, for example: the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthetase and the like. When the expression cassette is prepared, the various DNA fragments can be manipulated in order to provide the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. For this purpose, adapters or linkers can be used to join the DNA fragments or other manipulations can be used to provide convenient restriction sites, eliminate superfluous DNA, eliminate restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction digests, alignment, resubstitutions, for example transitions and transversions can be used.

As already indicated herein, the present invention provides vectors capable of expressing the genes of interest under the control of regulatory elements. In general, the vectors must be functional in plant cells. Sometimes, it may be preferable to have vectors that are functional in E. coli (for example, in the production of proteins to generate antibodies, analysis of DNA sequences, construction of inserts, obtaining quantities of nucleic acids). Vectors and methods for cloning and expression in E. coli are described in Sambrook et al. (supra). The transformation vector, comprising the promoter of the present invention operably linked to a nucleotide sequence isolated in an expression cassette, may also contain at least one additional nucleotide sequence corresponding to a gene that will be cotransformed in the organism. Alternatively, said sequence or additional sequences may be provided in another transformation vector. Vectors that are functional in plants can be binary plasmids derived from Agrobacterium. These vectors have the ability to transform plant cells. These vectors contain the sequences of the right and left borders necessary for their integration into the host (plant) chromosome. At least, between these sequences of the edges is the gene to be expressed under the control of the regulatory elements of the present invention. In one embodiment, a selectable marker and a reporter gene were also included. To facilitate obtaining sufficient amounts of vector, a bacterial origin allowing replication in E. coli can be used. Reporter genes can be included in the transformation vectors.

Examples of suitable reporter genes known in the art can be found, for example, in: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin was al. (Kluwer Academic Publishers), pgs. 1-33; DeWet I went to. (1987) Mol. Cell. Biol. 7: 725-737; Goff I went to. (1990) EMBO J. 9: 2517-2522; Kain I went to. (1995) BioTecgniques 19: 650-655; and Chiu went to. (1996) Current Biology 6: 325-330. Selectable marker genes can be included for the selection of transformed cells or tissues in the transformation vectors. These may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, for example: genes encoding resistance to chloramphenicol, Star Herrera et al. (1983) EMBO J. 2: 987-992; methotrexate, Herrera Estrella eí al. (1983) Nature 303: 209-213; Meijer eí al. (1991) Plant Mol. Biol. 16: 807-820; hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5: 103-108; Zhijian I went to. (1995) Plant Science 108: 219-227; streptomycin, Jones et al. (1987) Mol. Gen. Genet. 210: 86-91; Spectinomycin, Bretagne-Sagnard et al. (1996) Transgenic Res. 5: 131-137; bleomycin, Hille eí al. (1990) Plant Mol. Biol. 7: 171-176; sulfonamide, Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136; Bromoxynil, Stalker ef al. (1988) Science 242: 419-423; glyphosate, Shaw et al. (1986) Science 233: 478-481; phosphinothricin, DeBlock ei al. (1987) EMBO J. 6: 2513-2518. Other genes that could be useful in the recovery of transgenic events but that are probably not necessary in the final product include, for example: GUS (β-glucuronidase), Jefferson (1987) Plant Mol. Biol. Rep. 5: 387); GFP (green fluorescence protein), Chalfie et al. (1994) Science 263: 802; luciferase, Teeri eí al. (1989) EMBO J. 8: 343; and the corn genes that code for anthocyanin production, Ludwig et al. (1990) Science 247: 449. The transformation vector comprising the particular regulatory sequences of the present invention, operably linked to an isolated nucleotide sequence of interest in an expression cassette, can be used to transform any plant. In this way, plants, plant cells, plant tissue, seeds and similar genetically modified ones can be obtained. The transformation protocols may vary according to the type of plant or plant cell, ie monocotyledonous or dicotyledonous, intended for transformation. Methods of transforming suitable plant cells include microinjection, Crossway et al. (1986) Biotechniques 4: 320-334; electroporation, Riggs et al. (1986) Proc. Nati Acad. Sci. USA 83: 5602-5606; Agrobacterium-mediated transformation, see for example, Townsend et al. U.S. Patent No. 5,563,055; gene transfer, Paszkowski et al., (1984) EMBO. J. 3: 2717-2722) and ballistic acceleration of particles, see for example, Sanford et al. U.S. Patent No.: 4,945,050; Tomes eí al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe went to. (1988) Biotechnology 6: 923-926. See also Weissinger et al. (1988) Annual Rev. Genet. 22: 421-477; Sanford went to. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe I went to. (1988) Bio / Technology 6: 923-926 (soybean); Datta ef al. (1990) Biotechnology 8: 736-740 (rice); Klein went to. (1988) Proc. Nati Acad. Sci. USA 85: 4305-4309 (corn); Klein went to. (1988) Biotechnology 6: 559-563 (corn); Klein ef al. (1988) Plant Physiol. 91: 440-444 (corn); Fromm eí al. (1990) Biotechnology 8: 833-839; Hooydaas-Van Slogteren ef al. (1984) Nature (undres) 311: 763-764; Bytebier ef al. (1987) Proc. Nati Acad. Sci. USA 84: 5345-5349 (Liliaceae); From Wet ef to /. (1985) in The Experimental Manipulation of Ovule Tissues, ed. G. P. Chapman et al. (Longman, New York), pgs. 197-209 (pollen); Kaeppler ef al. (1990) Plant Cell Reports 9: 415-418; and Kaeppler ef al. (1992) Theor. Appl. Genet 84: 560-566 (fiber mediated transformation); D. Halluin ef al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li eí al. (1993J Plant Cell Reports 12: 250-255 and Christou et al. (1995) Anna / s of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (corn by Agrobacterium tumefaciens), the contents of which are incorporated herein by reference.The cells, which were transformed, can be grown to plants according to conventional manners, see, for example, McCormick et al. (1986) Plant Cell Reports, 5: 81-84.Then these plants can be grown and pollinated with the same transformed strain or with different strains and then the resulting progeny can be identified with the desired phenotypic characteristic.Two or more generations can be grown to ensure that the Preferential expression by seeds of the phenotypic characteristic of interest is maintained stably and is heritable EXAMPLES The present invention is further described with the following examples. illustrate the invention with reference to specific embodiments. These examples, while illustrating certain specific aspects of the invention, do not represent limitations or they circumscribe the scope of the described invention. It is evident that it is possible to effect certain changes and modifications within the scope of the appended claims. In the previous glossary, certain terms used in the present have been explained. All examples were carried out using standard techniques, which are well known and routine to those skilled in the art, except when otherwise described in detail. The routine molecular biology techniques of the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). All parts or amounts used in the following examples are by weight, unless otherwise specified. Unless otherwise indicated, the size separation of the fragments in the following examples was carried out using standard techniques of agarose and electrophoresis on polyacrylamide gel ("PAGE") of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and numerous other references such as, for example, Goeddel ef al., Nucleic Acids Res. 8: 4057 (1980). Unless otherwise described, ligations were performed using standard buffer solutions, standard incubation times and temperatures, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase. ("ligase") per 0.5 micrograms of DNA. Example 1: Construction of vector systems for the temporal and spatial expression with preference for seeds of the enzymes of the biosynthesis of cytoguins Construction of PHP 11466 v PHP 11467 v their cointegrates (PHP11551 and PHP11552 respectively). PPH 11466 and PHP 11467 were used in particle gun transformation protocols even when they contain the right and left borders for the tDNA. The versions denominated PHP11551 and PHP11552 were used in the transformation protocols mediated by Agrobacterium. The coding sequence of ipt was obtained as a BamHI / Hpal fragment of 732 bp and inserted into the expression cassette GLB1 (BamHI / Hpal, 4.9 kb) to obtain PHP11310. The corn GLB1 promoter (GenBank, Accession No. L22344, L22295) and the terminator (GenBank, Accession No. L22345, L22295) in PHP3303 comprise the expression cassette GLB1. The cassette pGLB1: ipt: GLB1 3 'was moved as two pieces (HindIII / BamHI 1401 bp and BamHI / EcoRI 1618 bp) in a T-DNA vector digested with EcoRI + HindIII (6.33 kb) to obtain PHP11363. Finally, a gene of a selectable marker (pUBI: UBIINTRON1: PAT optimized for corn: 35S 3 ') was added as a Hindlll fragment of 2.84 kb in PHP11363 digested with HindIII (9.35 kb). In PHP11466, the two genes are oriented in opposite ways to each other. In PHP11467, the two genes are oriented in the same direction. After a triparental conjugation, the cointegrated plasmid of PHP11466 / PHP10523 was named PHP11551. Also, the cointegrated PHP11467 / PHP10523 was named PHP11552.

Construction of PHP11404 v PHP11550 PHP 11404 was used with a transformation protocol mediated by biolistics. The plasmid has all the characteristics of the Agro version. The plasmid that was actually used with the Agro-mediated transformation protocols is PHP11550. The use of plasmid PHP9063 (pUBI: UBIINTRON1: ipt: pinll 3 '), allowed to create a restriction site Ncol in the start codon of ipt using site-directed mutagenesis (specifically, the set of MORPH ™ elements of 5 Prime -_ »3 Prime, Inc.). The resulting plasmid was named PHP11362. The ipt coding sequence was then moved as a Nol / Hpal fragment of 724 bp in PHP8001 (cut with BamHI, treated with Klenow to fill the superposition as a cohesive end which was then cut with Ncol, 4.9 kb) to obtain PHP11401 . PHP8001 contains the promoter and terminator GZ-W64A of the 27 KD zein gene from Z. mays (GenBank, Accession No. S78780). Then PHP11401 was digested with Pací + Kpnl and a 1.35 kb fragment was inserted into PHP11287 (digested with Pacl / Kpnl, 10.87 kb) to obtain PHP11404. PHP11287 is a T-DNA vector that already contains the pUBI construct: UBIINTRON1: PAT optimized for corn: 35S 3 'selectable marker, previously described. After a triparental conjugation the cointegrate of PHP11404 / PHP10523 was named PHP11550. Construction of PHP12975 The CIM1 promoter is described in U.S. Pat. No.: 09 / 377,648, filed August 19, 1999. Site-directed mutagenesis was used to create an Ncol site at the start of CIM1 translation (PHP12699). The promoter was cut out as a Sacl / Ncol fragment of 1.69 kb / Ncol and ligated to the ipt encoding sequence and the pinll terminator of PHP11362 to form the PHP12800. The C1M1 transcription unit: ipt: pinll was then moved as a 2.8 kb BstEII fragment in PHP12515 (9.5 kb) digested with BstEII, a binary vector that already contained the UBI construct: UBIINTRON1: MO-PAT: 35S selectable marker between the edges sequences. The resulting plasmid was named PHP12866. The triparental conjugation in LBA4404 of A. tumefaciens (PHP10523) allowed obtaining the cointegrated plasmid PHP12975. Construction of PHP12425 Plasmid PHP11404 (described above) was used as the initial plasmid to replace the GZ-W64A promoter with the LTP2 promoter from H. vulgare. The DNA of PHP11404 was digested with Notl and Kpnl (fragment of 9.46 kb) and separately with Ncol plus Kpnl (fragment of 1.24 kb). These two fragments were mixed with a 1.52 kb Notl / Ncol fragment of PHP8219 which contained the LTP2 promoter and ligated. The resulting plasmid product was named PHP12333. The triparental conjugation of this plasmid in LBA4404 of A. tumefaciens (PHP10523) allowed obtaining the cointegrated plasmid PHP12425. Triparental conjugation v selectable marker 35s: bar: pinll: All vectors were constructed using standard molecular biology techniques. The region of the T-DNA for transformation consists of the sequences of the T-DNA borders flanking a reporter gene and a selectable marker. The informant is inserted close to the right border of the T-DNA and consists of the 2.0 kb Pstl fragment of the Ubi-1 maize ubiquitin promoter (Christensen et al., 1992) with 5 'Hindlll and BamHI 3 flanking restriction sites. ' The ubiquitin promoter was ligated into the 5 'BamHI site of the beta-glucuronidase (GUS) reporter gene (Jefferson et al., 1986), which contains the second intron of potato ST-LS1 (Vancanneyt et al., 1990). The potato proteinase II (pinll) terminator (bases 2 to 310 of An et al., Plant Cell 1 (1): 115-122 (1989)) was ligated with 3 'cohesive ends with respect to the GUS coding sequence. . At the 3 'end of the terminator there is a Notl restriction site. The selectable marker consists of a 35S promoter of the improved cauliflower mosaic virus (bases -421 to -90 and -421 to +2 of Gardner, R.C., ef al., Nucí. Acids Res. 9: 2871-88 (1981)) with a flanking site Notl 5 'and Pstl 3'. A Pstl / Sall fragment containing the tobacco mosaic virus directive of 79 bp is inserted (Gallie, DR, et al., Nucí Acids Res. 15: 3257-73 (1987)) 3 'with respect to the promoter followed of a Sall / BamHI fragment containing the first corn dehydrogenase dehydrogenase intron ADH1-S (Dennis et al., 1984). The BAR coding sequence (Thompson, C.J., et al., Embo J. 6: 2519-23 (1987)) was cloned into the BamHI site, with the pinll terminator ligated in 3 '. The pinll signal is flanked by a 3 'Sacl site. The T-DNA of PHP8904 was integrated into the superbinary plasmid pSB1 (Ishida et al., 1996) by homologous recombination between the two plasmids. The HB101 strain of E. coli containing the PHP8904 with strain LBA4404 of Agrobacterium was mixed with pSB1 to create the co-integrated plasmid in Agrobacterium called LBA4404 (PHP10525) (by the method of Ditta, G., et al., Proc. Nati, Acad. Sci. USA 77: 7347-51 (1980)). LBA4404 (PHP10525) was selected for the resistance of Agrobacterium to spectinomycin and verified as recombinant by a SalI restriction digestion of the plasmid. Example 2: Corn transformation Biolistics: The polynucleotides of the invention contained in the vector are transformed into corn embryogenic callus by bombardment of particles, in general as described by Tomes, D. et al., In: Plant Cell, Tissue and Organ Culture: Fundamental Methods, Eds. O.L. Gamborg and G.C. Phillips, Chapter 8, pgs. 197-213 (1995) and is briefly described below. Transgenic maize plants were produced by bombardment of embryo-sensitive immature embryos with tungsten particles associated with plasmid DNA. The plasmids consist of a selectable and non-selectable structural gene. Preparation of particles: 15 mg of tungsten particles (General Electric), from 0.5 to 1.8, preferably from 1 to 1.8, and more preferably from 1, to 2 ml of concentrated nitric acid were added. This suspension is sonic at 0 ° C for 20 minutes (Branson Sonifier, Model 450, 40% output, constant duty cycle). The tungsten particles were pelleted by centrifugation at 10,000 rpm (Biofuge) for one minute and the supernatant was removed. Two milliliters of sterile distilled water was added to the pellet and a brief sonication was used to resuspend the particles. The suspension was pelleted, one milliliter of absolute ethanol was added to the pellet and a brief sonication was used to resuspend the particles. The particles were washed, pelleted and resuspended two more times with sterile distilled water, and finally the particles were resuspended in two milliliters of sterile distilled water. The particles were subdivided into aliquots of 250 ml and stored frozen. Preparation of the particle-plasmid DNA association: The stock solution of the tungsten particles was sonicated briefly in a sonicator on a water bath (Branson Sonifier, Model 450, 20% output, constant duty cycle) and 50 ml were transferred to a microcentrifuge tube. All the vectors were cis: ie the selectable marker and the gene of interest are on the same plasmid. These vectors were then transformed individually or in combination. The plasmid DNA was added to the particles for a final amount of DNA between 0.1 and 10 μg in 10 μl total volume and briefly sonicated. Preferably, 10 μg (1 μg / μl in TE buffer) of total DNA is used to mix DNA and particles for bombardment. Specifically, 1.0 μg of PHP 11404, 11466 and / or 11467 (1 μg / μl) was used, where any of the polynucleotides of the cytokinin biosynthesis enzymes can replace ipt, by bombardment. 50 microliters (50 μl) of 2.5 M sterile aqueous CaCl 2 was added and the mixture briefly sonicated and vortexed. 20 microliters (20 μl) of sterile 0.1 M aqueous spermidine was added and the mixture briefly sonicated and vortexed. The mixture was incubated at room temperature for 20 minutes with intermittent brief sonication. The particle suspension was centrifuged and the supernatant was removed. 250 microliters (250 μl) of absolute ethanol was added to the pellet, followed by brief sonication. The suspension was pooled into pellets, the supernatant was removed and 60 ml of absolute ethanol was added. The suspension is briefly sonic before loading the particle-DNA agglomeration onto macrocarriers. Tissue preparation Immature maize embryos of the High Type II variety are the target of the transformation mediated by bombardment of particles. East genotype is the F-? of two pure genetic lines, progenitors A and B, derived from the crossing of two known inbreds of corn, A188 and B73. Both parents were selected for their great competence in somatic embryogenesis, according to Armstrong et al., Maize Genetics Coop. News 65: 92 (1991). They crossed or crossed ears of Fi plants and the embryos were aseptically dissected from the developing caryopses when the scutellum just becomes opaque. This stage takes place approximately 9-13 days after pollination and more generally at 10 days after pollination approximately, depending on growth conditions. The embryos are 0.75 to 1.5 millimeters in length. The surfaces of the ears were sterilized with Clorox 20-50% for 30 minutes, followed by three washes with sterile distilled water. The immature embryos are cultivated with the scepter oriented upwards, on embryogenic induction medium comprising N6 basal salts, Eriksson vitamins, thiamin HCl 0.5 mg / l, sucrose 30 gm / l, L-proline 2.88 gm / l, 2,4-dichlorophenoxyacetic acid 1 mg / l, Gelrite 2 gm / l and AgNOß 8.5 mg / l. Chu et al., Sci. Sin. 18: 659 (1975); Eriksson, Physiol. Plant 18: 976 (1965). The medium is sterilized in an autoclave at 121 C for 15 minutes and placed in Petri dishes of 100 X 25 mm. AgNO3 was sterilized with filtration and added to the medium after the autoclave. The tissues were cultured in complete darkness at 28 ° C. After approximately 3 to 7 days, more usually 4 days, the embryo's scutellum swells to almost double its size and the protuberances on the surface of the scutellum coleorrhiza indicate the start of the embryogenic tissue. Up to 100% of embryos they present this response, but more commonly, the frequency of the embryogenic response is approximately 80%. When the embryogenic response is observed, the embryos are transferred to a medium composed of modified induction medium containing sucrose of 120 gm / l. The embryos are oriented with the pole of the coleorrhiza, the tissue that responds embryogenically, leaving the culture medium. Ten embryos were placed per Petri dish in the center of a Petri dish in an area approximately 2 cm in diameter. The embryos were maintained on this medium for 3-16 hours, preferably 4 hours, in complete darkness at 28 C just before bombardment with the particles associated with the DNA of the plasmids containing the selectable and non-selectable marker genes. For particle bombardment of the embryos, particle-DNA agglomerates are accelerated using a DuPont PDS-1000 particle acceleration device. The particle-DNA agglomeration was sonicated briefly and 10 ml was deposited on macrocarriers and the ethanol was allowed to evaporate. The macrocarrier is accelerated on a stainless steel stop screen by rupture of a polymer diaphragm (rupture disc). The rupture is effected by pressurized helium. The particle-DNA acceleration rate is determined on the basis of the breaking pressure of the rupture disk. Breaking disc pressures of 200 to 1800 psi are used, with 650 to 1100 psi being preferred and 900 psi being still more preferred. Multiple discs are used to apply a range of rupture pressures. The shelf containing the plate with the embryos is placed 5.1 cm below the base of the pallet of the macrocarrier (shelf No. 3). For him bombardment with particles of the immature cultivated embryos, a rupture disk and a macrocarrier with dried particle-DNA agglomerates were installed in the device. The pressure of He administered to the device is adjusted to 200 psi above the rupture pressure of the rupture disc. A Petri dish with the white embryos was placed in the vacuum chamber and placed in the projected path of the accelerated particles. Vacuum was created in the chamber, preferably approximately 28 mm Hg. Once the device was operated, the vacuum was released and the Petri box was removed. The bombarded embryos remain on osmotically adjusted medium during the bombardment and 1 to 4 days after the bombardment. The embryos are transferred to selection medium composed of basal N6 salts, Eriksson vitamins, thiamin HCl 0.5 mg / 1, sucrose 30 gm / l, 2,4-dichlorophenoxyacetic acid 1 mg / l, Gelrite 2 gm / l, AgNÜ3 0.85 mg / l and bialaphos 3 mg / l (Herbiace, Meiji). The bialaphos was added sterilized filtrate. The embryos were subcultured on freshly prepared selection medium at intervals of 10 to 14 days. After approximately 7 weeks, the embryogenic tissue, putatively transformed for both genes, from the selectable and non-selectable marker, proliferates from approximately 7% of the bombarded embryos. The putative transgenic tissue was rescued and said tissue derived from individual embryos is considered as an event and propagates independently on selection medium. Two propagation cycles were performed by cloning by visual selection of the smallest contiguous fragments of organized embryogenic tissue. A tissue sample from each event was processed to recover the DNA. The DNA is restricted with a restriction endonuclease and is subjected to testing with probes with primer sequences designed to amplify the DNA sequences that overlap with the plasmid portion of the cytokinin biosynthesis enzymes and that are not from the enzymes of cytokinin biosynthesis. The embryogenic tissue with the amplifiable sequence is allowed to advance towards the regeneration of the plant. To regenerate the transgenic plants, the embryogenic tissue is subcultured in a medium comprising MS salts and vitamins (Murashige &; Skoog, Physiol. Plant 15: 473 (1962)), myoinositol 100 mg / l, sucrose 60 g / l, Gelrite 3 g / l, zeatin 0.5 mg / l, indole-3-acetic acid 1 mg / l, cis-trans acid -abbscysic 26.4 ng / l and bialaphos 3 mg / l in Petri dishes of 100 X 25 mm and incubate in the dark at 28 ° C until the development of well-formed mature somatic embryos is observed. This requires approximately 14 days. Well-formed somatic embryos are opaque and cream colored and comprise identifiable scutellum and coleoptile. The embryos are individually subcultured in germination medium comprising MS salts and vitamins, myoinositol 100 mg / l, sucrose 40 gm / l and Gelrite 1.5 gm / l in Petri dishes of 100 X 25 mm and incubated with a photoperiod of 16 light hours: 8 hours dark and low 40 meinsteinsm "2sec" 1 of cold white fluorescent tubes. After about 7 days, the somatic embryos had germinated and produced well-defined shoots and roots. The individual plants are subcultured in germination medium in 125 X 25 mm glass tubes to allow the subsequent development of the plants. The plants are maintained with a photoperiod of 16 light hours: 8 dark hours and 40 meinsteinsm "2sec" 1 of white cold fluorescent tubes. After about 7 days, the plants are well established and transplanted to horticultural soil, softened, and placed in pots with a mixture of land for commercial greenhouse and let grow until reaching sexual maturity in greenhouse. An elite inbred line is used as a male element to pollinate the regenerated transgenic plants. Aarobacterium-mediated transformation: When an Agrobacterium-mediated transformation is used, the Zhao method is used as described in PCT Patent publication WO98 / 32326, the content of which is incorporated herein by reference. Briefly, immature maize embryos are isolated and the embryos contact a suspension of Agrobacterium (step 1: the infection step). In this step the immature embryos are preferably immersed in a suspension of Agrobacterium to initiate the inoculation. The embryos are co-cultivated for a time with Agrobacterium (step 2: the step of cocultivation). Preferably, the immature embryos are cultured on a solid medium after the Infection step. After this period of co-cultivation an optional step of "rest" is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for the plant transformants (step 3: resting step). Preferably, the immature embryos are cultured on solid medium with antibiotic, but without a selection agent, to remove the Agrobacterium and for the resting phase of the infected cells. Next, the inoculated embryos are cultured on medium containing a selection agent and the growing transformed calli are recovered (step 4: the selection step). Preferably, immature embryos are grown on medium solid with a selective agent resulting in the selective growth of the transformed cells. The calluses are then regenerated in plants (step 5: the regeneration step) and preferably the calluses growing on the selective medium are grown on solid medium to regenerate the plants. Example 3: Identification of transgenic corn lines rich in cytokinins The resulting transformants are evaluated for high levels of cytokinins using a combination of direct measurements and in vivo correlations. Viviparity experiments (glbl: ipt constructions): Given that seed dominance is considered to be controlled by the ratio of ABA: cytokinins, an elevated level of cytokinins in the seeds could induce the viviparous phenotype. Transformants Glb1:: ipt were initiated using GS3 embryos and transformation mediated by Agrobacterium (polynucleotides of the invention 11551 and 11552) or biolistic (polynucleotides of the invention .11466 and 11467). The seedlings were regenerated 2-3 months later and these seedlings (TO) were transferred to greenhouse after another 2-3 months. At the time of the anthesis, the TO plants were crossed with HG11 and viviparity was detected in the developing T1 seeds approximately 30 days later. Developing T1 seeds that exhibited the viviparous phenotype were rescued by replanting without drying the seeds. Viable plants were analyzed by PCR and a leaf identification was carried out to determine the presence of the ipt gene and the selectable marker (PAT gene). The T1 plants bloomed and the ears were autocrossed to generate T2 seeds. Those plants that contained the ipt gene (positive for PCR and identification of leaves) produced seeds that they secreted 3: 1 for the gene, while the negative plants for PCR and identification of leaves did not segregate. Determinations of cytokines: Ten seeds were harvested 19 and 23 days after pollination (DAP), each of four replications per event (11551 and 11552). The seeds were then separated into embryo and endosperm and harvested in liquid nitrogen. Each of the sampling days, embryonic tissue from all four replications was pooled and cytokinin levels determined. The endosperm tissue was processed in a similar manner. The results are shown in figure 1. Propagation of seeds glbl:: ipt: In order to propagate the viviparous seeds, half of the remaining plants of each event were harvested at 25 DAP. The ears were placed in drying boxes and ambient air (22 to 25 ° C) was passed through them to dry the seeds slowly. The drying boxes containing the transgenic ears were then transferred to a gr chamber and the seeds were dried to -12% humidity by passing air at 35 ° C through them for 3 to 5 days. Then the individual ears were peeled and the seeds were stored at 10 ° C and 50% RH. Determination of the phenotype: To determine the proportion of seeds that exhibit viviparity, the ears of the remaining half of plants were harvested approximately 45 DAP and the seeds were graded by the degree of viviparity. The four classes of viviparity were defined as: Class 1: No apparent swelling of coleoptile.

Class 2: Visible swelling of coleoptile, but without elongation. Class 3: Visible swelling of coleoptile with elongation after the scutellum, but without rupture of the pericarp. Class 4: Visible swelling of coleoptile with elongation after scutellum and rupture of the pericarp. The results are shown below in Table 1. TABLE 1 The results of the phenotypic evaluation showed that the presence of the ipt gene resulted in a higher incidence of viviparity (Classes 2 to 4), in relation to plants that do not contain the gene. Greater dry seed unit mass (constructions qz: ipf): Since the mass of the grains is a function of the number of cells and amyloplasts in the endosperm, and that the cytokinins have been related to a greater number of cells in the endosperm and in the differentiation of amyloplasts from the proplástidos, the seeds that exhibiting a higher level of cytokinins should show a corresponding increase in the dry unit mass of the seeds. Transformants Gz:: ipt were initiated using GS3 embryos and Agrobacterium-mediated transformation (polynucleotide of the invention 11550). The seedlings were regenerated in 2-3 months in 1997 and these seedlings (TO) were transferred to greenhouse after another 2-3 months. At the time of the anthesis, the TOs were crossed with HG11 and at the time of maturity the ears were harvested, peeled and seeds were used for further propagation of the seeds (both backcrossing with HG11 and self-pollination). Then T2 seeds were planted (both BC2 generation and autocross). The T2 plants were analyzed using PCR and leaf identification to determine the presence of the ipt gene and the selectable marker (PAT gene), respectively. Subsets of these plants were self-pollinated for cytokinin determinations or openly pollinated for phenotype determinations (yield and yield components). Cytokinin determinations: Samples can be collected and analyzed as follows. At 10, 16 and 22 DAP, between 50 and 100 seeds of two replications per event can be collected (each replication consisted of two subsamples) and the pedicel was removed. For 10 DAP samples, tissue can be placed remaining of the seeds directly in liquid nitrogen (tissue defined as "seed", composed primarily of pericarp, aleurone, endosperm and nucleus). By contrast, at 16 and 22 DAP, the embryo can be first dissected from the remnant seed tissue (tissue defined as "less embryo seed", and composed primarily of pericarp, aleurone and endosperm) and then both tissues are placed directly on the embryo. liquid nitrogen. Determination of the phenotype: To determine the effect of the gz:: ipt construction on the seed mass, individual plants were harvested manually at the time of physiological maturity (visible black layer), the seeds were peeled and dried in oven until reach constant mass (104 ° C, at least 3 days). The yield (g of plant) and yield components (ears per plant, seeds per ear and weight per seed) were determined in primary and secondary ears. Greater frequency of establishment of seeds and greater quantity of seeds (Construcciones Itp2: / pf): Since the yield is a combination of the frequency of establishment of seeds and number of seeds per ear, the seeds that present a higher level of cytokinins in The early stages of establishment and seed formation should come from ears with a corresponding increase in the establishment and quantity of seeds. Transformants Ltp2:: ipt were initiated using GS3 embryos and transformation mediated by Agrobacterium (12425). Seedlings were regenerated in 2-3 months in 1998 and these seedlings (TO) were transferred to greenhouse after another 2-3 months. At the time of the anthesis, the TOs were crossed with HG11 and at the time of maturity, the ears were harvested, peeled and seeds were used for additional seed propagation (both backcrossing with HG11 and self-pollination). The number of seeds per TO event and the number of events that established seeds were compared with the amount of other transgenic events with promoter: gene combinations other than Itp2: ipt. These are shown in Table 2. Table 2. Average seed set-up of ITp2 TO events: ipt gene compared to other genes in TO plants grown under conditions of Compared to the% seed establishment and the average number of seeds per TO plant, Itp2: ipt had both the highest% of TO plants that established seeds and the highest numerical average of seeds compared to six other transgenic combinations in TO plants grown at the same time and under the same greenhouse conditions. These results indicate that the expression of cytokinins in the aleurone layer of early seed development can increase yield because there is an increase both in the percentage of plants that establish seeds and in the amount of seeds established by cob. Subsequent generations will be grown in different field locations to determine the characteristics of seed establishment and seed quantity and seed yield compared to non-transgenic controls with the same genetic background. The levels of cytokinins in transgenic and non-transgenic grains of similar genetic background will also be measured. Cytokinin determinations: Samples can be collected and analyzed as follows. At 2, 6 and 22 DAP, between 50 and 100 seeds of two replications per event (each replication composed of two subsamples) can be collected and the pedicel removed. For samples of 2, 6 and 22 DAP, the remaining seed tissue can be placed directly in liquid nitrogen (the tissue is defined as "seed", composed primarily of pericarp, aleurone, endosperm and nucleus). EXAMPLE N ° 4: Isolation of ipt v isolation of ckx1-2 Briefly, PCR primers were constructed which preferably contain convenient restriction endonuclease sites. Two useful primers are shown below: SEQ ID N °: 38 (Upper primer with a Bam Hl site) 5'caucaucaucauggatccaccaatggatctacgtctaattttcggtccaac 3 'SEQ ID N °: 39 (Lower primer with a Hpal site) 5'cuacuacuacuagttaactcacattcgaaatggtggtccttc 3' The restriction sites entered are highlighted. The portion of the primer that binds to the annealed extends between nucleotides 22 and 19 to the 3 'terminus, respectively. A BamHI site was introduced "ggatcc" (highlighted) and a Kozak consensus sequence before the start codon and a Hpal "gttaac" site (also highlighted) after the stop codon. The manner in which the primers bind to the published sequence is schematically shown below. BamHI 5'caucaucaucauggatccaccaatggatctacgtctaattttcggtccaac aatggatctacgtctaattttcggtccaacttgcacaggaaagacatcgactgcgatagctcttgccca gcagactggcctcccagtcctctcgctcgatcgcgtccaatgctgtcctcaactatcaaccggaagcgggcga ccaacagtggaagaactgaaaggaacgactcgtctgtaccttgatgatcgccctttggtaaagggtatcattac agccaagcaagctcatgaacggctcattgcggaggtgcacaatcacgaggccaaaggcgggcttattcttga gggaggatctatctcgttgctcaggtgcatggcgcaaagtcgttattggaacgcggattttcgttggcatattattc gcaacgagttagcagacgaggagagcttcatgagcgtggccaagaccagagttaagcagatgttacgccc ctctgcaggtctttctattatccaagagttggttcaactttggagggagcctcggctgaggcccatactggaagg gatcgatggatatcgatatgccctgctatttgctacccagaaccagatcacgcccgatatgctattgcagctcga cgcagatatggagaataaattgattcacggtatcgctcaggagtttctaatccatgcgcgtcgacaggaacag aaattccctttggtgggcgcgacagctgtcgaagcgtttgaaggaccaccatttcgaatgtga 3'cctggtggtaaagcttacact cattgaucaucaucauc Hpal Agrobacterium strain was obtained tumefaciens containing the plasmid pTi Bo542 tumor inducing (See Guyon, P., et al., Agropine in null- type crow n gall tumors: Evidence for generality of the concept, Proceedings of the National Academy of Sciences (USA) 77 (5): 2693-97 (1980); Chilton, W.S., et al. Absolute stereochemistry of leucinopine, a crown gall opine, Phytochemistry (Oxford) 24 (2): 221-24 (1985); Strabala, T.J., et al., Isolation and Characterization of an ipt gene from the Ti plasmid Bo542, Molecular & General Genetics 216: 388-94 (1989)) and live bacteria were used for PCR annealing. Standard PCR conditions were used. An example of such conditions is the following: Reaction volume of 100 μl, with 0.5 μl of 10 ng / μl white plasmid, Taq Polymerase 0.05 units / μl, 0.5 μM of each of the primers, dNTP 0.8 mM, 1X buffer in a thin-walled tube. Mix the reagents, keep the mixture on ice. Add the white plasmid tube and then add the 100 μl of the reaction mixture to each tube. Pre-incubate in a thermal cycler at 95 ° C for 3 minutes. Then cycle five times at 95 ° C for 35 seconds, 55 ° C for 1 minute and 72 ° C for 1 minute. Continue with 30 cycles at 95 ° C for 35 seconds, 65 ° C for 1 minute and 72 ° C for 1 minute. Finish the reaction with an arrest for 10 minutes at 72 ° C and allow to soak at 6 ° C. Then clone the PCR product into DH5a cells using a set of Life Technologies elements according to the supplier's instructions. The DNA was extracted from putative transformants, < = _ cut with BamHI and Hpal and run on gel to confirm the transformation. This insert was purified on gel and transformed into a convenient expression vector, such as vector 7921 whose DNA contains an Ubi promoter and a pinll terminator. The preferred DNA sequence is provided in Molecular and General Genetics 216: 388-394 (1989). It contains an open reading frame that encodes a protein of 239 amino acid residues, with a deduced molecular weight of approximately 26.3 kDa (Calculated as the amount of amino acid residues X 110). Isolation of the corn cytokinin oxidase gene, cvtox 1-2 Another preferred DNA sequence is that shown below as SEQ ID NO: 1. It contains an open reading frame encoding a protein of approximately 535 amino acid residues, SEQ ID NO: 2, with a molecular weight deduced from approximately 58.9 kDa (Calculated as the amount of amino acid residues X 110). A copy of the cytokinin oxidase gene can be prepared by synthesis with the DNA synthesis protocols well known to those skilled in the art of gene synthesis. Alternatively, a copy of the gene can be isolated directly from the organism containing the cytokinin oxidase by PCR cloning. The corn cytokinin oxidase (ckxl) gene was cloned by Roy Morris of the University of Missouri and the sequence was deposited in Genbank. (Morris et al., 1999. Isolation of a gene encoding a glycosilated cytokinine oxidase from maize Biochem Biophys Res. Commun. 255 (2): 328-333 See also Houba-Herin et al., 1999. Cytokinine oxidase from Zea mays: purification, DNA cloning and expression in moss protoplasts, Plant J. (6): 615-626). PCR primers were constructed which conveniently contain suitable restriction endonuclease sites: Two useful primers are shown below: 5 'CATGCCATGGCGGTGGTTTATTACCTGCT 3' (with the Ncol site at the 5 'end) 5' CGGGATCCTCATCATCAGTTGAAGATGTCCT 3 '(with the BamHI site at the 3 'end) These primers were designed against the ckxl sequence and reverse transcriptase PCR (RT-PCR) was used to isolate the cytokinin oxidase genes from several different tissues of developing corn kernels. HE amplified DNA fragments from the following tissues: endosperms 10 DAP, 13 DAP, 18 DAP and 20 DAP; as well as embryos 10 DAP, 18 DAP and 20 DAP, where DAP means days after pollination. Fragments from all tissues migrated up to 1.6 Kb in the gel, which is the same value as for the published sequence. The authors selected one of the fragments (embryos 18 DAP) and sequenced the DNA. This fragment is called Cytox1-2 in the present and the full length sequence is shown below in SEQ ID NO: 1. At the amino acid level, there is 98% homology between the ckxl and cytox1-2 genes, so that the artisan will understand that cytox1-2 is a corn cytokininase oxidase gene. Example 5. Expression of transcripts in monocots A vector of a plasmid comprising the Zag2.1 promoter (SEQ ID No. 3) or the Zap promoter (SEQ ID No. 5, also known as ZmMADS) or the promoter was constructed. tb1 (SEQ ID NO: 17) operably linked to an isolated polynucleotide encoding ipt (SEQ ID NO: 1). This construction can be introduced into corn cells using the following procedure. Immature maize embryos from developing cariopses derived from the cross-linking of corn lines were dissected. The embryos were isolated 10 to 11 days after pollination when they measured between 1.0 and 1.5 mm in length. The embryos were then placed with the shaft side down and in contact with N6 medium solidified in agarose (Chu er al. (1975) Sci. Sin. Peking 18: 659-668). The embryos were kept in the dark at 27 ° C. From the scutellum of these immature embryos proliferate friable embryogenic calluses consisting of undifferentiated masses of cells with proembryoids and somatic embryoids on suspension structures. The calluses embryogenic isolated from the primary explant can be grown on N6 medium and subcultured on this medium every 2 to 3 weeks. Plasmid p35S / Ac (Hoechst Ag, Frankfurt, Germany), or an equivalent, can be employed in the transformation experiments in order to provide a selection marker. This plasmid contains the Pat gene (see European Patent Publication No. 0 242 236) which codes for phosphinothricin acetyl transferase (PAT). The PAT enzyme confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene, in p35S / Ac, is under the control of the 35S promoter of the cauliflower mosaic virus (Odell et al (1985) Nature 313: 810-812) and the 3 'region of the nopaline gene T-DNA synthase of the Ti plasmid of Agrobacterium tumefaciens. The particle bombardment method (Klein et al., (1987) Nature 327: 70-73) can be used to transfer genes to callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNA is added to 50 μl of a suspension of gold particles (60 mg per ml). Calcium chloride (50 μl of a 2.5 M solution) and spermidine free base (20 μl of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are centrifuged briefly (5 sec at 15,000 rpm) and the supernatant is removed. The particles are resuspended in 200 μl of absolute ethanol, recentrifuged and the supernatant is removed. Rinse again with ethanol and the particles are resuspended in a final volume of 30 μl of ethanol. An aliquot (5 μl) of the gold particles can be placed coated with DNA in the center of a Kapton frisbee (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic PDS-1000 / He equipment (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1,000 psi, a distance to the slit of 0.5 cm and a flight distance of 1.0 cm. For the bombardment, the embryogenic tissue is placed on filter paper on N6 medium solidified in agarose. The tissue is arranged as a thin layer and covers a circular area approximately 5 cm in diameter. The petri dish containing the fabric can be placed in the PDS-1000 / He chamber approximately 8 cm from the stop screen. The air in the chamber is then evacuated to a vacuum of 28 inches Hg. The macrocarrier is accelerated with a helium discharge wave using a rupture membrane that breaks when the He pressure in the discharge tube reaches 1,000 psi. Seven days after the bombardment the tissue can be transferred to N6 medium containing glufosinate (2 mg per liter) but without casein or proline. The tissue continues to grow slowly on this medium. After another 2 weeks the tissue can be transferred to fresh N6 medium containing glufosinate. After 6 weeks, areas of approximately 1 cm in diameter of actively growing calli can be identified on some of the plates containing the medium supplemented with glufosinate. These calluses can continue to grow when subcultured on the selection medium. Plants can be regenerated from transgenic callus by a first transfer of tissue clusters to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al, (1990) Bio / Technology 8: 833-839). Example 6. Expression of transgenes in dicots. Soy embryos are bombarded with a plasmid comprising the Zag2.1 promoter operably linked to a heterologous nucleotide sequence encoding ipt, as follows. In order to induce the formation of somatic embryos, cotyledons of 3-5 mm in length can be grown dissected from immature seeds, of sterilized surface of soybean cultivar A2872, with light or dark, at 26 ° C on an appropriate agar medium for 6-10 weeks. Somatic embryos that produce secondary embryos are trimmed and placed in a suitable liquid medium. After a repeated selection of the somatic embryo groupings that multiplied as embryos in the early globular stage, the suspensions are maintained as described below. The embryogenic soy suspension cultures can be maintained in 35 ml of liquid medium on a rotary shaker, 150 rpm, at 26 ° C with fluorescent light, with a program of 16: 8 hours of day / night. The cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium. The cultures in embryogenic soybean suspension can then be transformed by the particle gun bombardment method (Klein et al. (1987) Nature (London) 327: 70-73, US Patent No. 4,945,050 ). The DuPont Biolistic PDS1000 / HE (helium feedback) equipment can be used for these transformations. A selection marker gene that can be used to facilitate transformation in soybean is a transgene composed of the 35S promoter of the cauliflower mosaic virus (Odell et al (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene of the plasmid pJR225 (from E. coli; et al. (1983) Gene 25: 179-188) and the 3 'region of the nopaline synthetase gene of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette of interest, comprising the Zag2.1 promoter and a heterologous polynucleotide encoding ipt, can be isolated as a restriction fragment. This fragment can then be inserted into a single restriction site in the vector carrying the marker gene. To 50 μl of a suspension 60 mg / ml of 1 μm gold particles is added (in the order given): 5 μl of DNA (1 μg / μl), 20 μl of spermidine (0.1 M) and 50 μl of CaCl2 (2.5 M). The particle preparation is stirred for three minutes, passed through a microcentrifuge for 10 seconds and the supernatant is removed. The DNA coated particles are then washed once in 400 μl of 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA / particle suspension can be sonicated three times for one second at a time. Then, 5 μl of the gold particles coated with DNA are loaded onto each macrocarrier disk. Approximately 300-400 mg of a two week suspension culture is placed in an empty 60 x 15 mm Petri dish and the residual liquid is removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 tissue plates are bombarded. The membrane rupture pressure is defined at 1100 psi and the chamber is evacuated to a vacuum of 28 inches of mercury. The fabric is placed approximately 3.5 inches away from the retention screen and is bombarded three times. After From the bombardment, the tissue can be divided in half, placed back into the liquid and cultured as described above. Five to seven days after the bombardment, the liquid medium can be exchanged for fresh medium and eleven to twelve days after the bombardment by fresh medium containing 50 mg / ml hygromycin. This selective medium can be changed weekly. Seven to eight weeks after the bombardment, green transformed tissue can be seen growing from necrotic, non-transformed embryogenic clusters. The isolated green tissue is removed and inoculated in individual bottles in order to generate new embryogenic cultures transformed into suspension, propagated by cloning. Each new line can be treated as an independent transformation event. These suspensions can be subcultured and maintained as clusters of immature embryos or can be regenerated into whole plants by maturation and germination of the individual somatic embryos. Example 7. Analysis of the growth index of ears of T1 plants (hemicigotes D2F1) under non-stressing conditions The transformation of corn with the construction Zag2.1:: ipt was carried out as described in Example 5. The regenerated plants were pollinated with one of the progenitor genotypes to create seeds D2F1 (D2 refers to two doses of a parent, also known as T1 seed). Of the original 17 transformants, nine were selected to continue the evaluation based on favorable genetic complexity (ie, low to single copy number determined by Southern blot analysis), integrity of the plant transcription unit (determined by transfer analysis). Southern) and quantity adequate of seeds. The seeds D2F1 were planted according to a field study, well irrigated, with replications in Johnston, Iowa. The dry mass of the non-pollinated ears was measured when the emergence of the styles began and seven days later. The corn growth index (EGR) was calculated as the difference in the dry mass divided by the number of days. As shown in Figure 2, four of the nine events evaluated showed an increase in the growth rate of ears of corn, in relation to the sister plants negative for the transgene that were used as controls. The presence of the ipt transcript in developing ears representing the nine events was confirmed by RT-PCR. However, space restrictions in the field prohibited direct comparisons between plants positive for the transgene and plants negative for the transgene of the same event and of the same genotype. Instead, the control plants of this example were cultured from a pooled sample of segregated T1 seeds. The control lots were thinned to a standard density; In addition, plants positive for the transgene, identified by identification of leaves with a herbicide, were purified. As a result of the grouping of events and genotypes, and the variation in field conditions for transgenic plants vs. control, the differences in EGR between transgenic and control plants were mutated and the results were inconclusive. Therefore, the nine events were then used for the performance analysis the following year. Differences in the rate of corn growth are expected for transgenic events and can be evaluated appropriately by direct comparisons in which the genetic background and the field cultivation conditions are kept constant. Example 8. Analysis of the performance of T2 plants (D3F1 hemicigotes) under non-stressful conditions T2 seeds representing the nine selected events (derived from the pollination of T1 plants with a recurrent parent) were planted in a well irrigated field study. without replications, in Johnston, Iowa. The presence of the ipt transcript in developing ears was confirmed by Northern blot analysis. Negative sister plants were cultured for the transgene of each event as controls and a paired analysis of each event (a difference analysis) was carried out as well as an analysis of the average of the events. All plants of interest were emasculated and pollinated with a mixed non-transgenic male parent. Yield was determined by collecting primary ears. The grains were grouped by event and by the presence or absence of the transgene; the grains were then oven dried and the total dry mass was measured. The results are shown in Figure 3. The grain yield of seven of the nine events was greater than that of the controls. The number of grains, length of ears and grain mass were also measured; the results for the transgenics exceeded those of the non-transgenic sister plants in five of the nine events in length of the ears; and in five of nine events for grain quantity and dry matter per grain. Example 9. Analysis of homozygous plants D4F3 according to performance and height of the plants The progeny of the next generation of the nine selected events were evaluated in a field study, well irrigated, with replications, in Johnston, Iowa, in 2002. Sister plants negative for the transgene were planted as controls. All the plants of interest were emasculated; a mixture of non-transgenic plants served as a source of pollen. The height of the plants was measured in stages V10 and V12. (For questions about growth stages, see How a Corn Plant Develops, Iowa State University of Science and Technology, Special Report of the Cooperative Extension Service No. 48, June reprint, 1993.) Five of the nine events showed a statistically significant increase in the height of the plants, as shown in Figure 4. The yield was determined by collecting all the ears that contained grains. The grains were conveniently grouped, dried in the oven and the total dry mass was measured. As shown in Figure 5, three of the nine events showed a statistically significant increase in yield, including two of the events also showed a higher height of the plants. The number of ears, number of grains and mass of grains was also measured, as shown in Figure 6. Example 10. Analysis of yield, height of plants, leaf greenness, biomass and transkenetics expression of plants D4F3 under drought stress The homozygous progeny of the nine events under drought conditions were evaluated in a field study, with replications, in Woodland, California, in 2002. No supplemental irrigation was applied in order to generate a enough stress during the anthesis as to decrease the performance by 40% to 50%. To do this, the water was retained beginning at 920 GDU (units of degree of growth) after sowing and was restored at 1860 GDU. All plants of interest were emasculated and pollinated with a mixed non-transgenic male parent. The presence of the ipt transcript was determined by Northern blot analysis of developing stems, leaves and spikelets. The greenness of the leaves was measured approximately one week before flowering with a Minolta SPAD chlorophyll meter. The height of the plants was measured at the same time. Five of the nine events showed a statistically significant increase in the height of the plants, as shown in Figure 7. Four of these five events, and one additional event, showed greater greenness of the leaves, as shown in Figure 8. The yield was determined by collecting all ears with grains. The grains were conveniently grouped, dried in an oven and the total dry mass was measured. The number of grains, number of ears and mass of the grains were also measured. Three of the nine events showed better performance results, as shown in Figure 9; these three events also presented a greater height of the plants and greenery of the leaves. The increase in the biomass of the plants for one of these events is shown in Figure 10. In addition, in all the evaluated events, the plants positive for the transgene showed an increase in the stable state levels of the ipt transcripts in various tissues vegetative and reproductive in relation to that of plants negative for the transgene. Example 11. Analysis of the effect of transqen on low performance non-stressful conditions Numerous constructions were evaluated for their impact on performance in a preliminary examination at a location with supplementary irrigation as needed. All constructs were evaluated as multiple events, elite progenitor dose 2, and evaluated by their performance per se with two repetitions per event. Only plants positive for the transgene were harvested and then all the events were compared to each other for their performance advantages. The results are shown in Table 2, where the different constructions are classified by performance, higher performance in the upper part and lower performance in the lower part. The second column records the crude yield, while the third column records the difference between that input and the average of all the constructions.

Table 2 It can be seen that the four constructions in the upper part of the table presented a significantly higher performance than any of the other constructions evaluated. Similarly, three constructions exhibited a significantly lower performance than any of the other constructions in this test. The rest was not sufficiently distinguishable from each other and it can be assumed that their transgenes they create an obviously different impact simply from the basal genotype. In this test, there were four IPT constructions whose expression was addressed by Zag2.1 or Zap and this group represents the four highest performance constructs in this test: PHP19698 Zap:: IPT, PHP19020 Zag: ÍPT with Ubi: BAR, PHP19874 Zag: : IPT with 35s BAR (head-to-head) and original PHP15418 Zag:: IPT construction with 35s BAR (head-to-tail). When the rest of these constructions were evaluated with 10 events per construction, the retention of PHP15418 contained in fact more than 90 events. These results clearly show the impact of the coupling of the ipt gene with the promoter / regulatory sequence with the expression centered on the female meristems. Example 12. Analysis of the expression of zaq2: ipt in soybean Embryogenic soybean cultures were transformed in suspension with the particle bombardment method using the methods known in the art (Klein et al. (1987) Nature (London) 327: 70-73; U.S. Patent No. 4,945,050; Hazel, et al. (1998) Plant Cell. Rep. 17: 765-772; Samoylov, et al. (1998) In Vitro Cell Dev Biol.-Plant 34: 8-13). In particle gun bombardment methods it is possible to use the purified form of 1) whole plasmid DNA or 2) DNA fragments containing only recombinant DNA from the expression cassette (s) of interest. In this example, recombinant DNA fragments were isolated from the entire plasmid before use for bombardment. For every eight bombardments of soybean tissue, 30 μl of solution was prepared with 3 mg of 0.6 μm gold particles and up to 100 picograms (pg) of DNA fragment per base pair of the DNA fragment.

The soybean transformation experiments were carried out using two recombinant DNA fragments. The recombinant DNA fragment used to express the ipt gene was in a recombinant DNA fragment separated from the selectable marker gene that provided resistance to sulfonylurea herbicides. Both recombinant DNA fragments were co-precipitated on the gold particles. The stem tissue for these transformation experiments was obtained from immature soybean seeds. Secondary embryos were cut from the explants after 6 to 8 weeks on culture start medium. The starting medium was an MS solidified agar (Murashige and Skoog (1962) Physiol. Plant 15: 473-497) supplemented with vitamins, 2,4-D and glucose. Secondary embryos were placed in vials in liquid culture maintenance medium and maintained for 7-9 days on a rotary shaker at 26 +/- 2 ° C under a light intensity of -80 μEm-2s "1. crop maintenance was modified MS medium supplemented with vitamins, 2,4-D, sucrose and asparagine. Before the bombardment, tissue masses were separated from the flasks and moved to an empty 60 X 15 mm petri dish for bombardment. The tissue was dried with Whatman No. 2 filter paper. Approximately 100-200 mg of tissue corresponding to 10-20 masses (each 1-5 mm in size) per plate of bombardment tissue was used. After the bombardment, the tissue of each bombardment plate was divided and placed in two bottles with liquid culture maintenance medium per plate of bombardment tissue. Seven days after the bombing, the liquid medium in each bottle was replaced by maintenance of freshly prepared culture supplemented with selective agent 100 ng / ml (selection medium). For the selection of transformed soybean cells the selective agent used was a sulfonylurea compound (SU) with the chemical name, 2-chloro-N - ((4-methoxy-6-methyl-1, 3,5-triazin- 2-yl) aminocarbonyl) benzenesulfonamide (common names: DPX-W4189 and chlorsulfuron). Clorsulfuron is the active ingredient of DuPont's sulfonylurea herbicide, GLEAN®. The selection medium containing SU was replaced every week for 6-8 weeks. After the selection period of 6-8 weeks, islands of transformed green tissue growing from untransformed embryogenic necrotic clusters were observed. These putative transgenic events were isolated and maintained in SU media at a rate of 100 ng / ml for another 2-6 weeks with medium changes every 1-2 weeks to generate new embryogenic transformed cultures, propagated by cloning, in suspension. The embryos took a total of about 8-12 weeks in SU. Suspended cultures were subcultured and maintained as groups of immature embryos and were also regenerated in whole plants by maturation and germination of individual somatic embryos. 1400 T1 plants derived from the 42 transgenic events zag2 were grown in the greenhouse: ipt:: ALS with a high density (1400 plants in a space designed for 480). We used 18 plants of the variety 'Jack' grown in the same environment as controls. The plants were grown to maturity and then clusters of unusual pods or a higher pod load were visually selected. In eighty-three (83) plants zag2: ipt:: ALS and 18 Jack plants, the number of pods was measured by plant, number of seeds per plant and weight of seeds (converted to weight per 100 seeds). The data were subjected to ANOVA using the procedure PROC GLM in SAS and the separation of the means was completed using the PROC MEANS function of SAS. The null hypothesis evaluated consisted in determining if the zag2: ipt:: ALS plants selected visually by clusters of rare pods or greater load of apparent pods were significantly different from the non-transformed control (Jack). Plants of 34 events were selected and pod quantity data among all selected events were significantly different (p = 0.05) between the ipt and Jack plants. Among all the events, the selected zag2: ipt:: ALS plants presented an average of 32.1 pods, which was a significantly higher value (LSD = 5.2 pods) compared to the 25.4 pods that were the average of Jack. Five events were identified that were significantly different from Jack at the 0.05 level and at least 2 plants were measured from each event. When the pod data for these events were subjected to ANOVA, the zag2: ipt:: ALS plants were statistically different from the Jack plants. The plants of the 5 selected events presented an average of 42.3 pods, which was a statistically higher value (LSD = 5.9 pods) than the control. A count was made of the number of seeds of all the threshed plants of the two events with the highest average number of pods (AFS 3579.7.1 and AFS 3586.1.2). The Zag2: ipt:: ALS plants had an average of 73.6 seeds per plant, which was a significantly higher value (LSD = 11, 7 seeds) than the average amount of seeds per plant Jack (44.7 seeds) (Table 5). The events were not statistically different from each other. The seeds of each plant zag2 were weighed: ipt:: individual ALS and each individual Jack plant to determine if the size of the seeds was affected by the higher pod load. The weight of 10 seeds of individual plants of AFS 3579.7.1 and AFS 3586.1.2 was 16.6 grams, which was not statistically different (LSD = 1.1 gram) from the weight of 100 seeds of Jack control plants (16 , 2 grams). The data examined suggest that the construction zag2: ipt:: ALS could potentially affect the amount of pods and seeds per plant. In addition, the measured size of the seeds for the zag2: ipt:: ALS plants was not statistically different from the untransformed Jack control. A high level of variability was observed in the greenhouse environment; however, these preliminary data suggest that the zag2: ipt:: ALS construction can increase the retention of pods and seeds per plant without generating a statistical difference in the size of the seeds. Example 13: Isolation of eepl promoter sequences The procedure for isolating promoters is described in the User's Manual for the Universal Genome Walker element set marketed by Clontech Laboratories, Inc., Palo Alto, California. Genomic DNA was prepared by grinding leaves of 10-day seedlings of Zea mays in liquid nitrogen and the DNA was prepared using the DNeasy Plant element set (Qiagen, Valencia, California). The DNA was then used exactly as described in the Genome Walker User's Manual (Clontech PT3042-1, version PR68687). Briefly, the DNA was digested separately with the restriction enzymes Dral, EcoRV, Pvull, Seal and Stul, all to cut cohesive ends. In addition to the cohesive enzymes suggested by Clontech, three other cohesive enzymes, EcolCRI, Xmnl and Sspl, were also used in separate digestions. The DNA was extracted with phenol, then chloroform, then precipitated with ethanol. The Genome Walker adapters were linked by the ends of the restricted DNA, in order to create a "Genome Walker Library". To isolate the specific promoter regions, two primers (26-30 bp in length) specific to the gene, not overlapping, were designed that were complementary to the 5 'end of the maize genes identified from sequence databases. The primers were designed to amplify the 5 'region of the coding sequence, ie the 5' untranslated region and the promoter of the chosen gene. The sequences of the primers are shown below. A first round of PCR was carried out with each Genome Walker library with the AP1 primer from Clontech (SEQ ID N: 15) and the gene specific primer (gsp) 1 with the sequence shown in SEQ ID N0: 11. PCR was performed on an iCycler thermal cycler from Bio-Rad (Hercules, California) using the reagents supplied with the Genome Walker set of elements. The following parameters were used for the cycles: 7 cycles of 94 ° C for 2 seconds, then 68 ° C for 3 minutes, followed by 32 cycles of 94 ° C for 2 seconds and 67 ° C for 3 minutes. Finally, the samples were kept at 67 ° C for 4 minutes and then at 4 ° C for further analysis. As described in the User's Manual, the DNA of the first round of PCR and was used as an annealing in a second round of PCR using the AP2 primer from Clontech (SEQ ID N: 16) and the gene specific primer (gsp) 2 with the sequence shown in SEQ ID N °: 12. The parameters of the cycles for the second round were: 5 cycles of 94 ° C for 4 seconds, then 70 ° C for 3 minutes, followed by 20 cycles of 94 ° C for 4 seconds, then 68 ° C for 3 minutes. Finally, the samples were kept at 67 ° C for 4 minutes and then kept at 4 ° C. Approximately 10 ml of each reaction was run on a 0.8% agarose gel and the bands (usually 500 bp or more) were trimmed, purified with the Qiaquick gel extraction element set (Qiagen, Valencia, California) and were cloned into the TA pGEMTeasy vector (Promega, Madison, Wisconsin). The clones were sequenced for verification. The band produced from the Xmnl Genome Waiker library contained a sequence of 1.5 kb 5 'with respect to the specific primer of the gene in SEQ ID NO: 12. The eepl promoter region was obtained using the primers of SEQ ID. N °: 13 and 14, created from this sequence to amplify 1 kb of genomic DNA from the corn line A63. These primers added a Hindlll site at the 5 'end, a Ncol site at the start of translation and a 5' EcoRV site just from the Ncol site. These sites were added to help in the future construction of vectors. The PCR reaction was carried out with a iCycler thermal cycler from Bio-Rad (Hercules, CA) using the high fideiity PCR mix (Cat. No. 10790020, Invitrogen, Carlsbad, California). The following cycle parameters were used: 94 ° C for 2 seconds, followed by 30 cycles of 94 ° C for 20 seconds, 55 ° C for 30 seconds and 68 ° C for 1 minute. Finally, the samples were kept at 67 ° C for 4 minutes and then at 4 ° C for further analysis. The PCR products were then cloned into the vector pGEM-T Easy vector (Promega Corp. Madison, Wl). The clones were sequenced for verification. Example 14: Isolation of eep2 promoter sequences The procedure for isolating promoters is described in the Manual User for the Universal Genome Walker item set marketed by Clontech Laboratories, Inc., Palo Alto, California. Genomic DNA was prepared by grinding leaves of plants in step V6 of B73 of Zea mays in liquid nitrogen and the DNA was prepared using the set of elements to isolate PureGene DNA (Gentra Systems, Minneapolis, Minnesota). The DNA was then used exactly as described in the Genome Walker User's Manual (Clontech PT3042-1, version PR68687). Briefly, the DNA was digested separately with the restriction enzyme Dral, which generates cohesive ends. The DNA was extracted with phenol, then chloroform, then precipitated with ethanol. The Genome Walker adapters were linked by the ends of the restricted DNA, in order to create a "Genome Walker Library". To isolate the specific promoter regions, two primers (27 bp in length each) were designed, specific to the gene, not overlapping, which were complementary to the 5 'end of the maize genes identified from sequence databases. The primers were designed to amplify the 5 'region of the coding sequence, ie the 5' untranslated region and the promoter of the chosen gene. The sequences of the primers are shown below. A first round of PCR was carried out with each Genome Walker library with the AP1 primer from Clontech (SEQ ID N °: 15) and the specific primer of gene 1 (GSP1) with the sequence AAACACCTTCGGATATTGCTCCCTTTT (SEQ ID NO: 21). PCR was performed on a PTC-200 DNA Engine thermal cycler from MJ Research Inc. (Waltham, Massachusetts) using the reagents supplied with the Genome Walker kit. The following parameters were used for the cycles: 7 cycles of 94 ° C for 10 seconds, then 72 ° C for 3 minutes, followed by 32 cycles of 94 ° C for 10 seconds and 67 ° C for 3 minutes. Finally, the samples were kept at 67 ° C for 7 minutes and then at 8 ° C for further analysis. As described in the User's Manual, the DNA from the first round of PCR was diluted and used as a template in a second round of PCR using the Clontech AP2 primer (SEQ ID No. 16) and the gene-specific primer. 2 (GSP2) with the sequence TCTCGCATTTGCAGAAACGAACAACGT (SEQ ID N °: 22). The parameters of the cycles for the second round were: 5 cycles of 94 ° C for 10 seconds, then 72 ° C for 3 minutes, followed by 20 cycles of 94 ° C for 10 seconds, then 67 ° C for 3 minutes. Finally, the samples were kept at 67 ° C for 7 minutes and then kept at 8 ° C. Approximately 10 μl of each reaction was run on a 1.0% agarose gel and the PCR products of 500 bp or greater were trimmed, purified with the Qiaquick gel extraction element set (Qiagen, Valencia, California ). The band produced from the Dral Genome Walker library contained 1.0 kb of 5 'sequence with respect to the GSP2 primer and was cloned into the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, California). The clones were sequenced for verification. The eep2 promoter region was obtained by PCR from the plasmid using the primers corresponding to a 1027 bp 3 'region from the AP2 primer and 5' of the ATG start codon. The clones were sequenced for verification. The EST distribution for eep2 is as detailed below: o p0083.cldeu53r B73"Grain" "" "full grain 7 DAP" or p0124.cdbmq47r B73"Grain, Embryo" "" "embryo sac 6 days, Examined 1" or p0062.cymab46r B73"Grains, Endosperm" "" "embryo sacs (4 DAP)", or p0106.cjlps68r B73"Grain" "" "whole grains 5 DAP, examined 1" or p0124.cdbmq21 r B73"Grain, Embryo" "" Embryo sac 6 days, examined 1"or p0100.cbaab57r B73" Grain, Embryo, Endosperm "" "" Cenocitic embryo sacs (4 DAP), examined 1 (original bibl P0062) "or p0100.cbaac19r B73" Grain, Embryo, Endosperm "" "" Cenocitic embryo sacs (4 DAP), examined 1 (original bibl P0062) "or p0062.cymal89r B73" Grains, Endosperm "" "" cenocitic embryo sacs (4 DAP) ", or p0062.cymai74f B73"Grains, Endosperm" "" "embryo sacs (4 DAP) ", These data are very consistent with the limitation of the expression of this gene to developing seeds. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention is directed. All publications and patent applications are incorporated herein by reference as if each publication or individual patent application was specifically and individually incorporated herein by way of reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to provide greater clarity for its understanding, it is clear that certain changes and modifications are possible within the scope of the appended claims.

Claims (65)

1. CLAIMS: 1. A method for producing transgenic plants capable of regulating the expression of a cytokinin modulator gene in developing seeds or related female reproductive tissue, CHARACTERIZED BECAUSE: transforming plant host cells with a genetic construct capable of regulating temporally or spatially the expression of a cytokinin modulator gene in developing seeds or in related female reproductive tissue; and regenerating and recovering said transgenic plants.
2. 2. The method according to clause 1, CHARACTERIZED BECAUSE the transformation is carried out by a process selected from the group consisting of electroporation, PEG poration, particle bombardment, distribution with silicone fibers, microinjection and transformation mediated by Agrobacterium.
3. 3. The method according to clause 2, CHARACTERIZED BECAUSE said transformation process includes bombardment of particles.
4. 4. The method according to clause 2, CHARACTERIZED BECAUSE said transformation process is a transformation mediated by Agrobacterium.
5. 5. The method according to clause 1, CHARACTERIZED BECAUSE said genetic construction comprises a promoter that directs a temporal or spatial genetic expression in developing seeds or in the related female reproductive tissue, operatively linked to a cytokinin modulator gene. 6. The method according to clause 5, CHARACTERIZED BECAUSE said promoter is selected from the group formed by Zag2.1, zap, tb1, eepl, eep2,
6. F3.7, thxH, Zm40, ESR, PCNA2, led, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4 and ZmCkxd.
7. 7. The method according to clause 1, CHARACTERIZED BECAUSE the cytokinin modulator gene is selected from the group consisting of genes that encode cytokinin biosynthesis enzymes, cytokinin catabolic enzymes, cytokinin catabolic enzyme antagonists and enzyme agonists of cytokinins. biosynthesis of cytokinins.
8. 8. A transgenic plant, CHARACTERIZED BECAUSE it comprises a genetic construct stably integrated into the genome thereof, wherein said construct comprises a promoter operatively linked to a cytokinin modulator gene, where said promoter directs a temporal or spatial expression in seeds in development or in related female reproductive tissues of said plant.
9. 9. The plant according to clause 8, CHARACTERIZED BECAUSE said promoter is selected from the group formed by Zag2.1, zap, tb1, eepl, eep2, F3.7, thxH, Zm40, ESR, PCNA2, led, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4 and ZmCkxd.
10. 10. The plant according to clause 9, CHARACTERIZED BECAUSE the cytokinin modulator gene is selected from the group consisting of genes that encode enzymes of cytokinin biosynthesis, cytokinin catabolic enzymes, cytokinin catabolic enzyme antagonists and enzyme agonists of the cytokinins. biosynthesis of cytokinins.
11. 11. An isolated recombinant DNA, CHARACTERIZED BECAUSE it comprises a genetic construct that contains a promoter that directs a temporal or spatial genetic expression in developing seeds, or tissue female related player, operatively linked to a cytokinin modulator gene.
12. 12. Host cells, CHARACTERIZED BECAUSE they have stably incorporated in them the genetic construction of clause 11.
13. 13. A method to improve tolerance to stress and stability of performance in plants, CHARACTERIZED BY: transforming plant host cells with a genetic construct that preferentially directs the expression of a cytokinin modulator gene in developing seeds and related female reproductive tissue; and regenerating and recovering transformed plants from said cells.
14. 14. The method according to clause 13, CHARACTERIZED BECAUSE said preferential expression occurs between approximately 14 days before and approximately 25 days after pollination.
15. 15. The method according to clause 13. CHARACTERIZED BECAUSE said preferential expression occurs between approximately 0 and approximately 6 days after pollination.
16. 16. The method according to clause 13, CHARACTERIZED BECAUSE said preferential expression occurs between approximately 0 and approximately 12 days after pollination.
17. 17. The method according to clause 13, CHARACTERIZED BECAUSE said preferential expression occurs between approximately 4 and approximately 21 days after pollination.
18. 18. A transgenic plant, CHARACTERIZED BECAUSE it comprises a cassette of recombinant expression stably integrated in the genome of the same, where said cassette allows to increase the activity of cytokinins, where said transgenic plant presents a greater vigor without significant harmful effects on said greater activity of the cytokinins.
19. 19. Seeds, CHARACTERIZED BECAUSE they are from the transgenic plant of clause 18.
20. 20. The transgenic plant of clause 18, CHARACTERIZED BECAUSE said greater vigor is expressed in the presence or absence of abiotic stress.
21. 21. A method to develop a corn plant, CHARACTERIZED BECAUSE it uses the plant of clause 18 as a source of genetic material in a breeding program.
22. 22. The method of clause 21, CHARACTERIZED BECAUSE it also includes one or more techniques selected from the group consisting of: recurrent selection, mass selection, volume selection, backcrossing, pedigree, development of a synthetic and open pollination.
23. 23. The transgenic plant of clause 18, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a polynucleotide that encodes a protein involved in the biosynthesis of cytokinins.
24. 24. The transgenic plant of clause 18, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a polynucleotide encoding isopentenyl transferase.
25. 25. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the Zag2.1 promoter operably linked to a polynucleotide that encodes a protein involved in cytokinin biosynthesis.
26. 26. The transgenic plant of clause 23, CHARACTERIZED BECAUSE Said recombinant expression cassette comprises the eepl promoter operably linked to a polynucleotide that encodes a protein involved in the cytokinin biosynthesis.
27. 27. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the eep2 promoter operably linked to a polynucleotide that encodes a protein involved in cytokinin biosynthesis.
28. 28. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the zap promoter operably linked to a polynucleotide that encodes a protein involved in cytokinin biosynthesis.
29. 29. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the tb1 promoter operably linked to a polynucleotide that encodes a protein involved in cytokinin biosynthesis.
30. 30. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the ckx1-2 promoter operably linked to a polynucleotide that encodes a protein involved in cytokinin biosynthesis.
31. 31. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a promoter that directs a low level of constitutive expression of an operably linked polynucleotide that encodes a protein compromised in cytokinin biosynthesis.
32. 32. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises the F3.7 promoter.
33. 33. The transgenic plant of clause 23, CHARACTERIZED BECAUSE said recombinant expression cassette comprises (1) a promoter with preference for reproductive tissues operatively linked to a polynucleotide that encodes a protein involved in the biosynthesis of cytokinins and (2) one or more promoters or enhancing elements of a gene of great expression.
34. 34. The transgenic plant of clause 33, CHARACTERIZED BECAUSE the enhancer element comprises the 35S enhancer of the mosaic virus of the cauliflower.
35. 35. The transgenic plant of clause 34, CHARACTERIZED BECAUSE the 35S enhancer comprises SEQ ID NO: 4.
36. 36. The transgenic plant of clause 33, CHARACTERIZED BECAUSE the recombinant expression cassette comprises (1) the Zag2.1 promoter. operatively linked to a polynucleotide encoding ipt and (2) the 35S enhancer of the mosaic virus of the cauliflower.
37. 37. The transgenic plant of clause 36, CHARACTERIZED BECAUSE the recombinant expression cassette comprises (1) SEQ ID NO: 3 operatively linked to the coding region of SEQ ID NO: 1 and (2) SEQ ID NO: 4.
38. 38. The plant transgenic of clause 18, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a first polynucleotide engaged in the silencing of a gene that encodes a protein compromised in the decrease of the active cytokinin cluster.
39. 39. The transgenic plant of clause 38, CHARACTERIZED BECAUSE said protein compromised in diminishing the grouping of active cytokinins is the cytokinin oxidase.
40. 40. The transgenic plant of clause 38, CHARACTERIZED BECAUSE it also comprises a promoter that preferentially directs expression in developing seeds or in related female reproductive tissues, operatively linked to a second polynucleotide that encodes a protein involved in cytokinin biosynthesis.
41. 41. The transgenic plant of clause 38, CHARACTERIZED BECAUSE said first polynucleotide comprises an antisense sequence of a polynucleotide that encodes a protein committed to decrease the pool of active cytokinins.
42. 42. The transgenic plant of clause 38, CHARACTERIZED BECAUSE said first polynucleotide comprises a sequence effective to cosuppress a polynucleotide that encodes a protein compromised in decreasing the pool of active cytokinins.
43. 43. The transgenic plant of clause 38, CHARACTERIZED BECAUSE said first polynucleotide comprises an effective sequence in an RNAi of a polynucleotide that encodes a protein compromised in decreasing the pool of active cytokinins.
44. 44. A method of modulating the activity of cytokinins in a plant, where the modulated activity of cytokinins increases the vigor of the plants without significant harmful effects, CHARACTERIZED BECAUSE it comprises a stable transformation of said plant to result in an increase in the activity of cytokinins.
45. 45. The method of clause 44, CHARACTERIZED BECAUSE said plant is transformed in a stable manner with a cassette of recombinant expression able to increase the activity of cytokinins, where said transgenic plant It has a greater vigor without significant harmful effects with an increased activity of cytokinins.
46. 46. The method of clause 45, CHARACTERIZED BECAUSE said greater vigor is expressed in the presence or absence of abiotic stress.
47. 47. The method of clause 45, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a polynucleotide that encodes a protein involved in the biosynthesis of cytokinins.
48. 48. The method of clause 45, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a polynucleotide encoding isopentenyl transferase.
49. 49. The method of clause 45, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a promoter selected from the group consisting of Zag2.1, zap, tb1, eepl, eep2, F3.7, thxH, Zm40, ESR, PCNA2, led, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4 and ZmCkxd.
50. 50. The method of clause 45, CHARACTERIZED BECAUSE said recombinant expression cassette comprises (1) a promoter with preference for female reproductive tissues operably linked to a polynucleotide that encodes a protein involved in the biosynthesis of cytokinins and (2) one or more promoters or enhancing elements of a gene of great expression.
51. 51. The method of clause 50, CHARACTERIZED BECAUSE the enhancer element comprises the 35S enhancer of the mosaic virus of the cauliflower.
52. 52. The method of clause 51, CHARACTERIZED BECAUSE the 35S enhancer comprises SEQ ID NO: 4.
53. 53. The method of clause 50, CHARACTERIZED BECAUSE the recombinant expression cassette comprises (1) the bound Zag2.1 promoter. operatively to a polynucleotide encoding ipt and (2) the 35S enhancer of the mosaic virus of the cauliflower.
54. 54. The method of clause 53, CHARACTERIZED BECAUSE the recombinant expression cassette comprises (1) SEQ ID NO: 3 operatively linked to the coding region of SEQ ID NO: 1 and (2) SEQ ID N °: 4.
55. 55. The method of clause 45, CHARACTERIZED BECAUSE said recombinant expression cassette comprises a first polynucleotide engaged in the silencing of a gene that encodes a protein compromised in the decrease of the active cytokinin cluster.
56. 56. The method of clause 55, CHARACTERIZED BECAUSE said protein compromised in the decrease of the active cytokinin group is the cytokinin oxidase.
57. 57. The method of clause 55, CHARACTERIZED BECAUSE said plant is further transformed in a stable manner with a promoter that preferentially directs expression in developing seeds or in related female reproductive tissues, operatively linked to a second polynucleotide that encodes a protein involved in cytokinin biosynthesis.
58. 58. The method of clause 55, CHARACTERIZED BECAUSE said first polynucleotide comprises an antisense sequence of a polynucleotide that encodes a protein compromised in decreasing the active cytokinin cluster.
59. 59. The method of clause 55, CHARACTERIZED BECAUSE said first polynucleotide comprises a sequence effective to cosuppress the polynucleotide that encodes a protein compromised in the decrease of the grouping of active cytokinins.
60. 60. The method of clause 55, CHARACTERIZED BECAUSE said first polynucleotide comprises an effective sequence in the RNAi of a polynucleotide that encodes a protein compromised in the decrease of the active cytokinin cluster.
61. 61. An isolated promoter capable of directing transcription in a preferred manner in seeds, CHARACTERIZED BECAUSE said promoter comprises a nucleotide sequence selected from the group consisting of: a sequence comprising a fragment of the nucleotide sequence shown in SEQ ID No. 7 or 18; and the nucleotide sequence shown in SEQ ID N °: 7 or 18.
62. 62. The promoter isolated from clause 61, CHARACTERIZED BECAUSE it has the ability to direct transcription in a preferred manner in seeds, wherein said promoter comprises a fragment of the nucleotide sequence shown in SEQ ID NOS: 7 or 18. 63 The promoter isolated from clause 61, CHARACTERIZED BECAUSE it has the ability to direct transcription in a preferred manner in seeds, wherein said promoter comprises the nucleotide sequence shown in SEQ ID NOS: 7 or 18. 64. A recombinant expression cassette, CHARACTERIZED BECAUSE it comprises a promoter and a nucleotide sequence operatively linked to the promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a sequence comprising a fragment of the nucleotide sequence shown in FIG. SEQ ID N °: 7 or 18; Y the nucleotide sequence shown in SEQ ID N °: 7 or 18. 65. A plant stably transformed with an expression cassette, CHARACTERIZED BECAUSE it comprises a corn promoter and a nucleotide sequence operably linked to said promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a sequence comprising a fragment of the nucleotide sequence shown in SEQ ID NOS: 7 or 18; and the nucleotide sequence shown in SEQ ID N °: 7 or 18. MODULATION OF THE ACTIVITY OF CYTOCHININES IN PLANTS SUMMARY This invention relates in general to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for regulated expression in time and / or space of genes that affect the metabolically effective levels of cytokinins in plants, in particular in seeds and in female reproductive tissue related . This invention also relates to transgenic plants that have higher levels of cytokinin expression, where said transgenic plants exhibit characteristics that are useful, such as larger seed size, fewer abortions of younger grains, higher establishment of seeds under unfavorable environmental conditions or performance stability. The present invention also provides compositions and methods for regulating the expression of heterologous nucleotide sequences in a plant. The compositions comprise novel nucleotide sequences corresponding to promoters with preference for seeds, known as eepl and eep2. A method is also provided for expressing a heterologous nucleotide sequence in a plant using the promoter sequences described herein. The method comprises transforming a plant cell to contain a heterologous nucleotide sequence operably linked to one of the promoters of the present invention and regenerating a plant stably transformed from the transformed plant cell.