MXPA99000632A - Method of increasing growth and performance in plan - Google Patents

Abstract

The invention provides a method of producing a genetically modified plant, characterized by having increased growth and yield as compared to a corresponding wild-type plant which comprises increasing the level of cyclin expression in the plant. Genetically modified plants characterized by increased growth and yield are also provided.

Description

METHOD OF INCREASING GROWTH AND PERFORMANCE IN PLANTS Field of the Invention The present invention relates in general to the genetic design of plants, and specifically to a method for producing genetically engineered plants, characterized in that they have a higher growth and yield. BACKGROUND OF THE INVENTION For each plant species, there is a wide discrepancy in the growth of the plants, due to environmental conditions. Under most conditions, the maximum growth potential of a plant has not been realized. The reproduction of plants has shown that the resources of a plant can be redirected to individual organs to improve growth. Genetic engineering of plants, which involves the isolation and manipulation of genetic material, for example, DNA or RNA, and the subsequent introduction of that material into a plant or plant cells, has changed the reproduction of plants and agriculture considerably during recent years. Potentially higher values of crop feed, higher yields, feed value, reduced production costs, resistance to pests, tolerance to stress, drag resistance, the production of pharmaceutical products, chemical and biological molecules, as well as others are achieved. beneficial traits, through genetic engineering techniques. The ability to manipulate gene expression provides a means to produce new characteristics in transformed plants. For example, the ability to increase the size of a plant's root system allows it to have a greater assimilation of nutrients from the soil. Moreover, the ability to increase the growth of the leaves will increase the capacity of a plant to assimilate the solar energy. Obviously, the ability to control the growth of an entire plant, or specific target organs thereof, would be very desirable. SUMMARY OF THE INVENTION The present invention is based on the discovery that higher growth and yield in plants can be achieved by raising the level of cyclin expression. In a first embodiment, the invention provides a method for producing a genetically modified plant, characterized in that it has a higher growth and yield, compared to a corresponding wild-type plant. The method comprises contacting the cells of the plant with nucleic acid encoding a cyclin protein, wherein the nucleic acid is operably associated with a regulatory sequence, to obtain transformed plant cells; produce cells from the transformed plant cells; and selecting a plant that exhibits this increased yield. The nucleic acid encoding cyclin preferably encodes the cyclane cyclaAt. In another embodiment, the invention provides a method for producing a plant, characterized in that it has a higher yield, the method comprising contacting a plant with an agent that elevates the expression of cyclin above the expression of cyclin in a plant that does not have contact with the agent. The agent can be a transcription factor or a chemical agent that induces an endogenous cyclin promoter or another chemically inducible promoter that drives expression as the cyclin transgene. The invention also provides plants, plant tissue, and seeds produced by the methods of the invention. BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows continuous state levels of cdc2aAt mRNA and p34 protein, panel a; MRNA of cyclaAt during the induction of IAA of lateral root meristems, panel b; CyclaAt mRNA in selected non-induced transgenic lines, panel c; Normalized transcript levels are indicated relative to the wild type. Col-0, wild type; 1A2, 2A5, 4A3, 11A1: homozygous T2; 6A, 7A, 8A: heterozygous transgenic lines Ti. Figure 2 shows a hybridization analysis at the site of transcripts of cdc2aAt and cyclaAt in apices of roots and developing lateral roots. Panels A-D show cross-sections of passive roots (panels a.b) or primordial proliferating cells (panels c, d) that hybridized in anti-sense probes of cdc2aAt (a) or cyclaAt (b-d). Panels e, f show the abundance of cyclaAt mRNA in rows of contiguous meristematic cells in root apices. The accumulation of transcription is indicated by the deposit of silver grains, and is visualized by indirect red lighting. The scale bar is 10 microns in A-D, 5 microns in e. fc, accumulating the founding cell transcripts of cyclaAt; p, pericyclo cell layer; r, towards the apex of the ZC? Í Z-; S, towards the shoot. Figure 3 shows the fastest growth rate of the root in Arabidopsls thaliana (A. thaliana) that ectopically expresses the cyclane cyclaAt. Panel a, wild type (left) or transgenic line 6A (generation TI) that contains the fusion of the cdc2 gene to At:: cyclaAt (right). Arabidopsis seeds were coated on MS agar (3 percent sucrose), and cultured in a vertical orientation for 7 days. The plants transformed with the vector alone or with the unrelated promoter :: uidA constructs or with a cdc2aAt:: cyclaAt fusion where the leader not translated 5 'of cdc2aAt was interrupted by a transposon insert DS, did not show this phenotype. Panel b, wild type (left) or transgenic line 6A (TI generation) (right) 6 days after the induction of IAA of the lateral roots. Hydroponically cultured one-week-old plants were treated with 10 μM of IAAeff to stimulate the development of lateral roots. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides ods for increasing the yield of a plant, such as an agricultural crop, by raising the level of cyclin expression in the plant. Expression of increased cyclin in the plant cells competent to divide results in increased growth of the plant. In a preferred embodiment, the invention provides a od for producing a genetically modified plant, characterized in that it has a higher yield, compared to a plant that has not been genetically modified (for example, a wild-type plant). The od comprises contacting the cells of the plant with nucleic acid encoding a cyclin protein, wherein the nucleic acid is operably associated with a regulatory sequence to obtain transformed plant cells; produce plants from the transformed plant cells; and then select a plant that exhibits the highest growth and yield. As used herein, the term "yield" or "yield of the plant" refers to a higher growth of the crop, and / or to a higher biomass. In one embodiment, a higher yield results from a higher growth rate and a larger root size. In another modality, a higher yield is derived from shoot growth. In yet another embodiment, a higher yield is derived from the growth of the fruit. The term "genetic modification", as used herein, refers to the introduction of one or more exogenous nucleic acid sequences, eg, cyclin coding sequences, as well as regulatory sequences, into one or more plant cells, that can generate viable, sexually competent whole plants. The term "genetically modified", as used herein, refers to a plant that has been generated through the aforementioned process. The genetically modified plants of the invention are capable of self-pollination or having a cross-pollination with other plants of the same species, in such a way that the foreign gene, carried in the germ line, can be inserted in, or reproduced in, varieties of agriculturally useful plants. The term "plant cell", as used herein, refers to protoplasts, ga producing cells, and cells that regenerate into whole plants. As used herein, the term "plant" refers to either a whole plant, a plant part, a plant cell, or a group of plant cells, such as a plant tissue or a seed of plant. Also included are plants within the meaning of "plant". The plants included in the invention are any plants susceptible to transformation techniques, including gymnosperms and angiosperms, both monocotyledonous and dicotyledonous. Examples of monocotyledonous angiosperms include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye, oats, and other cereal grains. Examples of dicotyledonous angiosperms include, but are not limited to, tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cabbage crops or Brassica oleracea (for example, cabbage, broccoli, cauliflower, briquella collections), radish, carrots, beets, eggplants, spinach, cucumber, chayote, melons, varieties of melons, sunflowers, and different ornamentals. Examples of wood species include poplar, pine, redwood, cedar, oak, and so on. The term "exogenous nucleic acid sequence", as used herein, refers to a foreign nucleic acid for the host host plant, or native to the plant if the native nucleic acid is substantially modified from its original form. For example, the term includes a nucleic acid that originates from the host species, wherein this sequence is operably linked to a promoter that differs from the wild type or natural promoter. In the broad method of the invention, at least one nucleic acid sequence encoding cyclin is operably linked to a promoter. It may be desirable to introduce more than one copy of the cyclin polynucleotide into a plant for better expression of cyclin. For example, multiple copies of a cyclin polynucleotide would have the effect of increasing cyclin production even more in the plant. The term "regulatory sequence", as used herein, refers to a nucleic acid sequence that can control the transcription of an operably associated gene. Accordingly, the placement of a gene under the regulatory control of a promoter or regulatory element means the placement of the gene in such a way that the expression of the gene is controlled by the regulatory sequences. In general, the promoters are placed 5 '(upstream) of the genes they control. Accordingly, in the construction of promoter gene combinations, the promoter is preferably placed upstream of the gene, and at a distance from the transcription initiation site that approximates the distance between the promoter and the gene that it controls in the establishment natural. As is known in the art, some variation in this distance can be tolerated without losing the function of the promoter. In a similar manner, the preferred placement of a regulatory element, such as an enhancer, with respect to a heterologous gene placed under its control, reflects its natural position in relation to the structural gene that it regulates naturally. The nucleic acids encoding cyclin used in the present invention include nucleic acids encoding mitotic cyclins such as, for example, cyclin B; nucleic acids encoding S-phase cyclins, such as, for example, cyclin A, and nucleic acids encoding Gl phase cyclins. Specific cyclins that can be used herein include cylaAt, cyc3aAt, cycdl, cycd2, and the like. Preferably, the nucleic acid used in the method of the invention encodes the cyclaAt protein (Accession number of Genebank X62279). The genetically modified plants of the present invention are produced by contacting a plant cell with a nucleic acid sequence encoding the desired cyclin. To be effective once introduced into the plant cells, the nucleic acid encoding cyclin must be operably associated with a promoter that is effective on the cells of the plant, to cause transcription of the cyclin transgene. Additionally, a polyadenylation sequence or a transcription control sequence, also recognized in the cells of the plant, can also be employed. It is preferred that the nucleic acid be introduced by means of a vector, and that the vector harboring the nucleic acid sequence also contain one or more selectable marker genes, such that the transformed cells can be selected from non-target cells. transformed into culture, as described herein. The term "operably associated" refers to the functional link between a regulatory sequence, preferably a promoter sequence, and the nucleic acid sequence encoding cyclin regulated by the promoter. The operably linked promoter controls the expression of the cyclin nucleic acid sequence. The expression of the cyclin genes used in the present invention can be driven by a number of promoters. Although the endogenous or native promoter of a structural gene of interest can be used for the regulation of transcription of the gene, preferably the promoter is a foreign regulatory sequence. When it is desired to increase growth and yield in the whole plant, cyclin expression should be directed to all cells in the plant that are capable of dividing. This can be done using an active promoter in all meristems. These promoters include, for example, the cdc2a promoter and the cyc07 promoter. (See, for example, Ito et al, Plant Mol. Biol. 2.4: 863, 1994, Martinez et al, Proc.Nal.l Acad.Sci.U.A., 8.9: 7360, 1992, Medford et al., Plant Cell,. 3: 359, 1991; Terada et al., Plant Journal, 3: 241, 1993; issenbach et al., Plant Journal, 1: 411, 1993). When it is desired to increase growth and yield in a specific organ, the expression of cyclin should be directed towards the appropriate meristem, for example, shoot meristem, floral meristem, root meristem, and so on. This can be done using a tissue-specific promoter. Examples of tissue-specific promoters active in shoot meristems are described in Atanassova et al., Plant Journal, 2: 291, 1992, and Medford et al., Plant Cell, 3: 359, 1991. Examples of specific promoters. of active tissue in the floral meristems are the promoters of the genes agamous and apétala 1, and are described in Bowman et al., Plant Cell, 3: 149, 1991; and Mandel et al., Na ture, 360: 273, 1992. The particular promoter selected should be capable of causing sufficient expression of cyclin, to cause a higher yield and / or a higher biomass. It should be understood that cyclin expression can be altered in cells that are competent to divide. The promoters used in the vector constructions of the present invention can be modified, if desired, to affect their control characteristics. Optionally, a selectable marker can be associated with the nucleic acid encoding cyclin. As used in this, the term "marker" refers to a gene encoding a trait or a phenotype that allows the selection of gum or the screening of a plant or plant cell containing the marker. Preferably, the marker gene is an antibiotic resistance gene, wherein the appropriate antibiotic can be used to select the transformed cells from among the cells that are not transformed. Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin B-phosphotransferase, thymidine kinase, xanthine-guanine phospho-ribosyltransferase, and 3'-O-phosphotransferase II amino-glycoside. Other suitable markers are known to those skilled in the art. To begin a transformation process in accordance with the present invention, it is first necessary to construct a suitable vector, and introduce it appropriately into the plant cell. The vectors employed in the present invention for the transformation of a plant cell include a nucleic acid sequence encoding cyclin operably associated with a promoter. The details of the construction of the vectors used herein are known to those skilled in the art of plant genetic engineering. The nucleic acid sequences encoding cyclin used in the present invention can be introduced into plant cells using Ti plasmids from Agrobacterium tumefa ciens (A. tumefa ciens), root-inducing plasmids (Ri) from Agrobacterium rhizogenes (A. rhizogenes), and plant virus vectors. (For reviews of these techniques see, for example, Weissbach & amp;; Weissbach, 1988. Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pages 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2nd edition, Blackie, London, Chapters 7-9, and Horsch et al., Science, 227: 1229, 1985, both incorporated herein by reference). In addition to the transformation vectors of plants derived from the Agrobacterium Ti or Ri plasmids, alternative methods may involve, for example, the use of liposomes, electroincorporation, chemicals that increase the recovery of free DNA, transformation using viruses or pollen, and the use of microprojection. One skilled in the art will be able to select an appropriate vector for introducing the nucleic acid sequence encoding cyclin in a relatively intact state. Accordingly, any vector that produces a plant carrying the nucleic acid encoding introduced cyclin should be sufficient. Even the use of a naked piece of DNA would be expected to confer the properties of this invention, albeit at a low efficiency. The selection of the vector, or if a vector is used, is typically guided by the selected transformation method. The transformation of plants according to the invention can be carried out essentially in any of the different ways known to those skilled in the art of plant molecular biology. (See, for example, Methods Enzymology, Volume 153, 1987, Wu and Grossman, Eds., Academic Press, incorporated herein by reference). As used herein, the term "transformation" means the alteration of the genotype of a host plant, by introducing the cyclin nucleic acid sequence. For example, a nucleic acid encoding cyclin can be introduced into a plant cell using A. tumefaciens containing the Ti plasmid, as mentioned briefly in the above. In the use of a culture of A. tumef ciens as a transformation vehicle, it is more convenient to use a non-oncogenic strain of Agrobacterium as the vector carrier, in such a way that a normal non-oncogenic differentiation of the transformed tissues is possible. It is also preferred that the Agrobacterium host a binary Ti plasmid system. This binary system comprises: 1) a first Ti plasmid having a virulence region essential for the introduction of transfer DNA (T-DNA) into the plants, and 2) a chimeric plasmid. The chimeric plasmid contains at least one boundary region of the T-DNA region of a wild-type Ti plasmid flanking the nucleic acid to be transferred. It has been shown that binary Ti plasmid systems are effective in the transformation of plant cells (De Framond, Biotechnology, .1: 262, 1983, Hoeke a et al., Nature, 303: 179, 1983). This binary system is preferred because it does not require integration into the Ti plasmid of A. tumefa ciens, which is an older methodology. Methods involving the use of Agrobacterium in the transformation in accordance with the present invention include, but are not limited to: 1) Agrobacterium cocultivation with cultured isolated protoplasts; 2) transformation of plant cells or tissues with Agrobacterium; or 3) transformation of seeds, apices, or meristems with Agrobacterium. In addition, a genetic transfer can be carried out by a transformation in the plant by Agrobacterium, as described by Bechtold et al. (C. R. Acad. Sci. Paris, 316: 1194, 1993), and as exemplified in the examples of this. This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells. The preferred method for introducing a nucleic acid encoding cyclin into plant cells is to infect these plant cells, an explant, a meristem, or a seed, with transformed A. tumefa ciens, as described above. Under appropriate conditions known in the art, the transformed plant cells are grown to form suckers, roots, and further grown in plants. ~ Alternatively, the nucleic acid encoding cyclin can be introduced into a plant cell using mechanical or chemical elements. For example, the nucleic acid can be mechanically transferred to the plant cell by microinjection using a micropipette. Alternatively, the nucleic acid can be transferred to the plant cell through the use of polyethylene glycol, which forms a precipitation complex with the genetic material that is recovered by the cell. The nucleic acid encoding the cyclin can also be introduced into the plant cells by electroincorporation (Fromm et al., Proc.Nal.l Acad.Sci.E.U.A., 82 .: 5824, 1985, which is incorporated in FIG. the present as a reference). In this technique, plant protoplasts are electroenhanced in the presence of vectors or nucleic acids containing the relevant nucleic acid sequences. The electrical impulses of a high field strength permeabilize the membranes reversibly, allowing the introduction of nucleic acids. The electroincorporated plant protoplates reform the cell wall, divide, and form a plant callus. The selection of transformed plant cells with the transformed gene can be done using phenotypic markers as described herein. Another method for introducing a nucleic acid encoding cyclin into a plant cell, is high-speed ballistic penetration with small particles with the nucleic acid to be introduced into or inside the matrix of these particles, or on its surface ( Klein et al., Na ture 327: 70, 1987). Methods of bombardment transformation are also described in Sanford et al. (Techniques 3: 3-16, 1991) and Klein et al. (Bio / Techniques 10: 286, 1992). Although normally only one introduction of a new nucleic acid sequence is required, this method particularly provides multiple introductions. Cauliflower mosaic virus (CaMV) can also be used as a vector for introducing nucleic acid into plant cells (U.S. Patent No. 4,407,956). The genome of the CaMV viral DNA is inserted into a bacterial plasmid of origin, creating a recombinant DNA molecule, which can be propagated in bacteria. After cloning, the recombinant plasmid can be cloned again, and can be further modified by introduction of the desired nucleic acid sequence. The modified viral portion of the recombinant plasmid is then separated from the bacterial plasmid of origin, and used to inoculate the plant cells or plants. As used herein, the term "contacting" refers to any means for introducing a nucleic acid encoding cyclin into a plant cell, including chemical and physical means as described above. Preferably, contacting refers to introducing the nucleic acid or the vector containing the nucleic acid into the plant cells (including an explant, a meristem, or a seed), by means of A. tumefaciens transformed with the nucleic acid which codes for cyclin, as described above. Normally a plant cell is regenerated to obtain an entire plant from the transformation process. The immediate product of the transformation is referred to as a "transgenote". The term "growth" or "regeneration", as used herein, means growing an entire plant from a plant cell, a group of plant cells, a plant part (including seeds), or a piece of plant (for example, from a protoplast, callus, or part of tissue). Regeneration from protoplasts varies from species to plant species, but in general a suspension of protoplasts is first made. In certain species, the formation of the embryo can then be induced from the protoplast suspension. The culture medium will generally contain different amino acids and hormones, necessary for growth and regeneration. Examples of the hormones used include auxins and cytokinins. Efficient regeneration will depend on the medium, the genotype, and the history of the crop. If these variables are controlled, the regeneration is reprossable. Regeneration also occurs from calluses, explants, organs, or parts of plants. The transformation can be carried out in the context of a regeneration of organs or parts of plants. (See Methods in Enzymology, Volume 118, and Klee et al., Annual Review of Plant Physiology, 38: 467, 1987). Using the leaf disc transformation regeneration method of Horsch et al., Science, 227: 1229, 1985, discs are grown on a selective medium, followed by shoot formation in about 2 to 4 s. The shoots that develop are separated from the calluses and transplanted to an appropriate selective root inducer medium. The rooted plants are transplanted to the ground as soon as possible after the roots appear. The seedlings can be put back in pot, as required, until they reach maturity. In vegetatively propagated crops, mature transgenic plants are propagated using cuttings or tissue culture techniques to produce multiple identical plants. A selection of desirable transgenotes is made, and new varieties are obtained and propagated in a vegetative manner for commercial use. In seed-propagated crops, mature transgenic plants can auto-cross to produce a homozygous inbred plant. The resulting inbred plant produces seeds that contain the newly introduced foreign genes. These seeds can be grown to produce plants that produce the selected phenotype, for example, a higher yield. The parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, roots, fruit, and the like, are included in the invention, in the understanding that these parts comprise plant cells that have been transformed as described. Also included are progeny and variants, and mutants of regenerated plants, within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences. Plants that exhibit higher growth and / or yield, compared to wild-type plants, can be selected by visual observation. The invention includes plants produced by the method of the invention, as well as plant tissue and seeds. In another embodiment, the invention provides a method for producing a plant, characterized in that it has a higher growth and yield, by contacting a plant that may have a higher yield, with an inducing amount of the cyclin promoter of an agent that induces expression. of the cyclin gene. Induction of cyclin gene expression results in the production of a plant that has a higher yield, compared to a plant that has not made contact with the agent. A "plant that can have a higher yield" refers to a plant that can be induced to express its endogenous cyclin gene to achieve a higher yield. The term "promoter-inducing amount" refers to the amount of an agent necessary to elevate the expression of the cyclin gene above the expression of cyclin in a plant cell that has not made contact with the agent, by stimulating the promoter. of endogenous cyclin. For example, a transcription factor or a chemical agent can be used to elevate the expression of the gene from the native cyclin promoter, thereby inducing the promoter and expression of the cyclin gene. The invention also provides a method for providing enhanced transcription of a nucleic acid sequence in a selected tissue. The method comprises cultivating a plant having integrated in its genome, a nucleic acid construct comprising an exogenous gene that encodes a cyclin protein, this gene being operably associated with a specific tissue by which the transcription of this gene is increased in the selected tissue. The development of the plant is plastic with the post-embryonic organogenesis mediated by the meristems (Steeves and Sussex, Pa t terns in Plant Developmen t, 1-388 (Press Syndicate of the University of Cambridge, Cambridge, 1989)). Although cell division is intrinsic to meristem initiation, maintenance, and proliferative growth, the role of the cell cycle in the regulation of growth and development is unclear. To resolve this question, we examined the expression of the cdc2 and cyclin genes, which encode the catalytic and regulatory subunits, respectively, of the cyclin-dependent protein kinases that control the progress of the cell cycle (Murray and Hunt, The Cell Cycle (New York), 1993). Unlike cdc2, which is expressed not only in apical meristems, but also in passive meristems (Martinez et al, Proc. Na tl. Acad. Sci. USA, j3_9: 7360, 1992), transcripts of cyclaAt were accumulated specifically in active meristems, and dividing cells immediately before cytokinesis. The ectopic expression of cyclaAt under the control of the cdc2aAt promoter in Arabidopsis plants, markedly accelerated growth without altering the pattern of development or without inducing neoplasia. Accordingly, the expression of cyclin is a limiting factor for growth. The foregoing description generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. EXAMPLE 1 A full length cyclain cyclin cDNA was placed under the control of the cdc2aAt Arabidopsis promoter (Hemerly et al., 1992, supra). The chimeric gene was cloned into a T-DNA transformation vector bearing the hygromycin phosphotransferase selection marker (Hyg1), and transformed into Arabidopsis using the infiltration method at • empty (Bechtold and Pelletier, Acad Sci. Paris, Life Sci., 316: 1194, 1993), to introduce Agrobacterium tumefaciens. Several independent transgenic lines that had high steady-state levels of cyclaAt mRNA showed a dramatic increase in both root and lateral root growth rates, correlated with fresh weight, dry mass, and proportionally increased DNA content, but not the cell size. The best growth was ordered, without differences in morphology, and clearly it was not neoplastic. Arabidopsis seedlings (ecotype Col umbia) were grown in 20 milliliters of MS medium (Murashige and Skoog, Physiol. 15 Plan t. , 15 .: 473, 1962). Plants from 8 to 10 days of age were transferred to an MS medium regulated with 50 mM potassium phosphate, pH 5.5, and root initiation was stimulated • by addition of IAA up to 10 μMeff (IAA not dissociated). The roots were collected at the indicated time points, and 20 the total RNA and the protein were isolated. 500 nanograms of poly (A) + RNA were separated on 1 percent formaldehyde gels (Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, New York, 1987), were transferred to Nytran membranes ( Schleicher and Schüll), 25 and hybridized in 32 P-labeled probes corresponding to nucleotides (nt) 674-1004 of cyclaAt (Hemerly et al., Proc. Na tl.Acad.Sci. EUA, _3_9: 3295, 1992), or to nucleotides 661-1386 of cdc2aAt from Arabidopsis (Hirayama et al., Gene, 105: 159, 1991), followed by hybridization with nucleotides 2576-2824 of UBQ3 from Arabidopsis (Norris et al., Plan t Mol. Biol., 2J1: 895, 1993) for normalization The spots were quantified with a Molecular Dynamics Phosphorimager, and the cyclaAt is a single-copy gene in Arabidopsis. The total protein was separated on 12 percent SDS-PAGE, and transferred to PVDF membranes. 24cdc2a t was detected with serum elevated in rabbits against the peptide YFKDLGGMP (SEQ ID NO: 1), corresponding to amino acids 286-294, and visualized by Improved Chemiluminescence Assay (Amersham). Figure 1 shows the continuous state levels of cdcSaAt mRNA and protein p34, panel a; of cyclaAt mRNA during the induction of IAA of lateral root meristems, panel b; of cyclaAt mRNA in selected non-induced transgenic lines, panel c; Normalized transcript levels are indicated relative to the wild type. Col-0, wild type; 1A2, 2A5, 4A3, 11A1: homozygous T2; 6A, 7A, 8A: Ti heterozygous transgenic lines. CyclaAt mRNA levels in lines 4A3, 6A, 7A, 8A, and 3A exceeded those of the wild-type roots induced by IAA. The levels of the cdc2 mRNA and the p34cdc2 protein per cell did not change markedly following the stimulation of the lateral root initiation by indole acetic acid (IAA) (Figure 1, panel a). Therefore, although the expression of cdc2 is correlated with the competence to divide, the root growth and the initiation of the lateral roots do not seem to be limited by the abundance of the p34 catalytic subunit of cyclin-dependent protein kinase, and, moreover, the ectopic expression of cdc2 in transgenic Arabidopsis failed to disrupt growth growth (Hemerly et al., EMBO J., 14: 3925, 1995). In contrast, treatment with IAA of Arabidopsis roots induced the expression of several cyc genes from low basal levels, and in particular of cyclaAt mRNA, which encodes a mitotic cyclin (Hemerly et al., Supra), exhibited a rapid increase in 15 to 20 times (Figure 1, panel b). Figure 2 shows an in situ hybridization analysis of transcripts of cdcSaAt and cyclaAt in root apices and in developing lateral roots. The a-d panels show cross sections of passive roots (panels a, b), or primordial proliferating cells (panels c, d) that were hybridized in anti-sense probes of cdc2aAt (a) or cyclaAt (b-d). Panels e, f, show the abundance of cyclaAt mRNA in rows of contiguous meristematic cells at the root apices. The accumulation of transcription is indicated by the deposit of silver grain, and visualized by indirect red lighting. The scale bar is 10 microns in a-d, 5 micras in e, fc, and the founding cell accumulated transcripts of cyclaAt; p, cell layer of the pericycle; r, towards the apex of the root; s, towards the offspring. Tissue samples were processed for hybridization at the site in order to examine the expression of cyclin transcripts. The samples were treated with IAA 10M. After 8 or 24 hours of incubation, radish roots (Rhapanus sa tiva var. Scarlet Globe) were processed as described (Drews et al., Cell, 65: 991, 1991). Sections (8 micras) were hybridized in an RNA probe labeled with 33p, corresponding to nucleotides 674-1004 of cyclaAt (Hemerly et al., Supra) (Figure 2, panels be), or a probe labeled with 35S was used, corresponding to nucleotides 661-1386 of cdc2aAt (Hirayama et al., supra), for 14 hours at 48 ° C in 50 percent formamide. After hybridization, the final washings were for 1 hour at 58 ° C in 0.015 M NaCl, and then the plates were exposed for 3 weeks (cyclaAt) or 5 days (cdcSaAt). After development, the silver beads were illuminated laterally with red light, the samples were visualized by phase contrast, and double exposures were taken on FUJI Velvia film. The images were assembled in ADOPE Photoshop. For the analysis summarized in Figure 2, panel f, the silver beads were counted and the cell size was measured in the row of cells shown in Figure 2, panel e.

Hybridization at the site showed that, unlike cdc2, transcripts of cyclaAt were not detected in the passive cells of the pericycle, but accumulated in the simple cytoplasmically dense cells of the incipient lateral root primordia, and in the emerging organ was expressed cyclaAt exclusively in the meristem (Figure 2, panels ad). Moreover, the cruciferous roots consisted of rows of long cells that were presented by transverse divisions followed by longitudinal expansion (Dolan et al., Developmen t, 119: 71, 1993), and within this contiguous spatial deployment of cellular division phases in sequencing, transcripts of cyclaAt accumulated only in large cells immediately before cytokinesis, declining to background levels in small adjacent fixed cells (Figure 2, panels e, f). A similar rigorous spatio-temporal relationship of cyclin expression and mitosis was observed in the apical meristems of Antirrhinum shoots (Fobert et al., EMBO J., 13: 616, 1994). The close correlation between the expression of cyclaAt and cell division during root apical meristem growth and lateral root initiation, together with the cyclaAt promoter activity pattern inferred from the expression of cyclaAt:: uidA gene fusions in transgenic Arabidopsis (Ferreira et al., Cell, 6: 1763, 1994), suggested that the abundance of cyclin could be a key factor regulating the growth and development of the root. To test this hypothesis, transgenic Arabidopsis (Bechtold and Pelletier, Acad Sci Paris, Life Sci., 316: 1194, 1993) containing cyclaAt under the control of the cdc2aAt promoter were generated. Five transformants were obtained in which the mRNA level of cyclaAt in the untreated roots exceeded that observed in the roots stimulated with IAA of the wild type plants (Figure 1, panel C), and these lines were selected for another study. A Nhel site was introduced into the third codon of the cyclaAt cDNA by in vitro mutagenesis, and this open reading frame was subsequently ligated to the cdc2aAt promoter with an Xbal site generated in vi tro at codon 3. This fragment was ligated into pBiB- Hyg (Becker et al., Pl. Mol. Biol., 20: 1195, 1992), and transfected into Agrobacterium tumefaciens GV3101 (Koncz and Schell, Mol.Gen. Genet., 204: 383, 1986). Arabidopsis thaliana (A. thaliana) (ecotype Columbia) was transformed by vacuum infiltration (Bechtold et al., Supra), and the transgenic seedlings (TO generation) were selected on MS plates containing 30 micrograms / milliliter of hygromycin. 52 independent transgenic lines were obtained, and high levels of cyclaAt mRNA were detected in 9 of the 11 lines analyzed in detail. Growth assays were performed on the heterozygous IT and homozygous T2 progeny, as indicated. Figure 3 shows the highest root growth rate in Arabidopsis that ectopically expresses the cyclane cyclaAt. Panel a, wild type (left) or transgenic line 6A (generation Ti) containing the genetic fusion cdc2aAt:: cyclaAt (right). Arabidopsis seeds were coated on agar (3 percent sucrose), and cultured in a vertical orientation for 7 days. Plants transformed with the vector alone, or with the unrelated promoter :: uidA constructs, or with a cdc2aAt:: cyclaAt fusion where the untranslated 5 'leader of cdc2aAt was interrupted by a DS transposon insert, did not show this phenotype Panel b, wild type (left), or transgenic line 6A (TI generation) (right) 6 days after induction with IAA of the lateral roots. Hydro-organically cultured one-week-old plants were treated with 10 μM IAAeff to stimulate lateral root development. A strong expression of the cdc2aAt:: cyclaAt transgene caused a marked increase in the rate of organized growth of the root (Figure 3, panel a). Homozygous or heterozygous seeds were coated on MS agar, and the plants were grown in a vertical orientation for 7 days with a program of 16 hours of day / 8 hours of night at 22 ° C. Four images of each plate were acquired with a Speedlight Platinum frame fastener (Lighttools Research) at 24-hour intervals, and the root growth was analyzed with an NIH-Image by measuring the displacement of the root apices. Following the growth analysis, the roots of 10 plants of each class were collected, and RNA was analyzed. To measure cell sizes, the roots were released by incubation overnight in saturated chloral hydrate, visualized with Normarski optics, photographed, and analyzed with NIH-Image. The statistical analysis (test and with unpaired variations) was done with MS Excel. Root growth in the plants treated with IAA is evaluated 3 and 6 days after the induction, by determining the fresh weight of the separated roots of the plants grown in liquid, and then the dry weight followed by lyophilization during 24 hours. Total DNA was extracted from the dried material (Ausubel et al., Supra). In the heterozygous T2 progeny, the highest growth rate, as measured by the apex displacement of the main root in a time-lapse photograph, was strictly cosegrouped with the expression of the transgene and the individuals lacking the transgene grew at the same rate than wild-type plants (Table 1). The average size of the epidermal, cortical, endodermal, and pericycle cells was equivalent or slightly reduced in the cdc2aAt:: cyclaAt transformants, compared with the wild type plants (Table 2), and therefore, the highest growth reflects the highest number of cells instead of cell size. The pattern of spontaneous lateral root initiation, and the overall morphology of the root, was indistinguishable in the wild-type and transgenic plants (Figure 3, panel a). When treated with 1 μM IAA, which induces a well separated lateral root primordia, the frequency of the primordia initiated per unit length of the main roots was not altered (average of 1.08 initials / millimeter with a standard deviation of 0.09 in the wild type, comparing with 1.14 +/- 0.07 and 1.09 +/- 0.13 in the two transgenic lines examined). However, the growth and development of the lateral roots followed by the induction by 10 μM IAA was markedly accelerated in the cdc2aAt:: cyclaAt transformants, giving rise to a much more extensive root system (Figure 3, panel b). The best root growth in the cdc2aAt:: cyclaAt plants following the treatment with IAA, superficially resembles the alfl phenotype (Celenza et al., Genes &; Development, 9.:2131, 1995), and these plants have high levels of cyclaAt transcripts, but in contrast to the cdc2aAt:: cyclaAt transformants, alfl plants initiate supernumerary side roots. The greater gain of several times the fresh weight in the cdc2aAt:: cyclaAt plants treated with IAA, compared to the equivalent wild-type controls, was accompanied by a marked increase in DNA content and dry weight (Table 3). The confocal microscope confirmed that the best growth response to IAA, which was also observed in several lines that showed a weaker expression of cdc2aAt:: cyclaAt, did not reflect the cell vacuolation stimulation or elongation. Therefore, ectopic cyclin expression improves root growth by stimulating the cell division activity in the meristems, thereby increasing the speed of cell production without altering the meristem organization. The above data indicate that the expression cdc2aAt:: cyclaAt is sufficient to improve growth from the apical meristems established, suggesting that cell cycle activity regulates meristem activity. However, the failure to induce free organ primordia by ectopic expression of cyclaAt under the control of the cdc2aAt promoter, implies additional control points in the generation of a new apical meristem, either through a regulation after the translation of cyclin-dependent protein kinase activity, or the operation of parallel regulatory trajectories. In most animal cells, the commitment to cell division occurs late in Gl (Pardee, AB, Science, 246: 603, 1989), and cyclin DI and cyclin E are rate-limiting for the progress of Gl in cultured cells (Ohtsubo and Roberts, Science, 259: 1908, 1993; Quelle et al., Genes Dev., 7: 1559, 1993; Resnitzky and Reed, Mol. Cell Biol., 15.:3463, 1995). Elevated levels of cyclin DI are observed in several tumors (Motokura et al, Na ture, 350: 512, 1991; Rosenberg et al, Proc.Nal.l. Acad.Sci.U.A., 88 .: 9638, 1991; Withers et al. Mol. Cell Biol., 11: 4864, 1991), and ectopic expression in transgenic mice promotes hyperplasia and adenocarcinomas (Wang et al., Na ture, 369: 669, 1994). In contrast, the ectopic expression of cyclaAt did not result in neoplasia, but it stimulated organized growth, without altering the organization of the meristem or its size, as was monitored by the confocal microscope. Moreover, the morphology of the transgenic plants was not altered, and the greater growth was accompanied by an accelerated organ development. Accordingly, the expression of cyclin is a crucial limiting factor upstream in an intrinsic regulatory hierarchy that regulates meristem activity, organized growth, and indeterminate development of the plant. This regulatory hierarchy, which is distinctly different from that of animals, where particular development limits proliferative growth, exemplified by the strict morphogenetic control of cell division during muscle differentiation (Halevy et al., Science, 267: 1018, 1995 Skapek et al., Science, 267: 1022, 1995), can underline the surprising plasticity of plant growth and development (Drew, MC Ne Phytol., 7j3: 479, 1975). Cyclin abundance can function as a rheostat to allow for flexible growth control in response to changes in the environment, such as nutrient availability.

Table 1 Apical Root Growth Table 1 shows a comparison of apical root growth rates. Plant line = independent transformants (except Col-0). (+) = plants that show a better growth phenotype due to the presence of adequate levels of nucleic acid encoding cyclin. (-) = plants that have lost the introduced cyclin-encoding nucleic acid, or that do not exhibit sufficient cyclin expression for better growth. Speed = speed of displacement of root apex per unit of time. (* denotes significantly different values of the growth rate of the wild type). n = number of individual plants analyzed.

Table 2 Table 3 Growth of the Plantita Root System Table 2 shows a comparison of cell size; and Table 3 shows a comparison of root growth after treatment with IAA, in wild type and transgenic Arabidopsis lines containing the cdc2aAt:: cyclaAt genetic fusion. Lines 3A, 6A, 7A, 8A are heterozygous IT populations with more than one introduced transgene; (+) denotes plants with higher levels of transcription of cyclaAt, (-) plants with wild type cyclaAt transcription levels. The following T2 lines are homozygous for cdc2aAt:: cyclaAt: 2A5, 4A3, and 11A1; expression of constitutive cyclaAt in 4A3, but not in 2A5 and 11A1, which exceeds the wild-type levels induced with IAA (Figure 1). n, number of plants analyzed. *, means that they are significantly different from the wild type; for a, P < 0.001, for b, P < 0.01. Fresh weight = weight of the freshly cut root system. Dry weight = weight after 24 hours of drying. The above description of the invention is exemplary for purposes of illustration and explanation. It should be understood that different modifications can be made without departing from the spirit and scope of the invention. In accordance with the above, it is intended that the following claims be construed to cover all these modifications.

Claims (1)

1. REIVI DICACIO ES 1. A method of producing a genetically modified plant, characterized by increased growth and yield compared to the wild-type plant 5, said method comprising: contacting plant cells with nucleic acid encoding a cyclin protein, wherein said nucleic acid is operatively associated with a regulatory sequence, to obtain transformed plant cells; produce plants from said transformed plant cells; and selecting a plant that exhibits said increased yield. 2. The method of claim 1, wherein the plant 15 genetically modified exhibits increased root growth. 3. The method of claim 1, wherein the genetically modified plant exhibits increased shoot growth. 4. The method of claim 1, wherein the cyclin is cycllaAt. 5. The method of claim 1, wherein the regulatory sequence is a promoter. 6. The method of claim 5, wherein the promoter 25 is selected from the group consisting of constitutive promoters and inducible promoters. The method of claim 1, wherein the contacting is by physical means. 8. The method of claim 1, wherein the contacting is by chemical means. The method of claim 1, wherein the plant cell is selected from the group consisting of protoplasts, gamete producing cells, and cells that are regenerated into whole plants. The method of claim 1, wherein said nucleic acid is contained in a vector derived from T-DNA. 11. A plant produced by the method of claim 1. 12. Plant tissue derived from a plant produced by the method of claim 1. 13. A seed derived from a plant produced by the method of claim 1. 14. A method of producing a plant characterized by having increased growth and yield, said method comprising contacting a plant with an agent that elevates cyclin expression on the expression of cyclin in a plant not in contact with the agent. 15. The method of claim 14, wherein the increased growth and yield are a result of increased root growth. 16. The method of claim 14, wherein the increased growth and yield result from increased shoot growth. 17. The method of claim 14, wherein the cyclin is cycllaAt. 18. The method of claim 14, wherein the agent is a transcription factor. 19. The method of claim 14, wherein the agent is a chemical agent. 20. A method of providing enhanced transcription of a nucleic acid sequence in plant tissue, wherein said method comprises: culturing a plant having integrated into its genome a nucleic acid constructing element comprising nucleic acid encoding a cyclin protein, wherein said nucleic acid is operatively associated with a tissue-specific promoter with which the expression of said nucleic acid encoding cyclin is increased in said plant tissue.