MXPA00008525A - Transgenic crops accumulating fructose polymers and methods for their production - Google Patents

Abstract

A method for producing fructose polymers of various lengths throug expression of plant-derived FTF genes in transgenic monocot plants is disclosed. Also disclosed are transgenic monocot plants seeds derived from said plants wherein the level of fructan that accumulates in the cells of the transgenic monocot plants and seeds is increased when compared to the level of fructan that accumulates in the cells of monocot plants and that do not contain the instant chimeric gene(s) encoding plant-derived FTF genes.

Description

TRANSGENIC CROPS THAT ACCUMULATE FRUCTOSE POLYMERS? METHODS FOR YOUR PRODUCTION FIELD OF THE INVENTION The present invention relates to methods for the synthesis and accumulation of fructose polymers in transgenic maize [Zea mays L.) by the selective expression of fructosyl transferase genes derived from plants.

TECHNICAL BACKGROUND The higher plants accumulate several commercially useful carbohydrate polymers, such as starch and fructan. Starch and cellulose are currently used in numerous food and non-food applications in their native form, but are more likely to be modified enzymatically or chemically, which greatly expands their usefulness.

Fructans are linear or branched polymers of repeated fructose residues. The number of residues contained in an individual polymer, also known as the degree of polymerization (GP), varies widely depending on the source from which it is isolated. For example, REF .: l-18-3 fructan synthesized by fungal species, as in Aspergillus syndowi could contain only two or three fructose residues. In contrast, polymers with a GP of 5000 or greater are synthesized by several bacterial lines, including Bacillus amyloliquefaciens and Streptocuccus mutans. Fructans of intermediate size, with a GP of 3 to 60, are found in almost 40,000 species of plants (Science and Technology of Fructans, (1993) M. Suzuki and N. Chatterton, eds CRC Press Inc., Boca Raton, FL, pp. 169-190).

Regarding size, fructose polymers are not metabolized by humans. Because of this, and because of its softness, small fructans with a GP of 3-4 are used in a wide variety of low-calorie food products. The size of the polymer is critical with respect to its commercial use. The high GP polymers are not smooth, however, they provide texture to food products very similar to that of fat. The high GP fructan used as a fat substituent also contributes very little to the caloric value of the product.

Fructans are also considered to be an excellent source of fructose for the production of syrup of superior fructose (Fuchs, A. (1993) in Sci en ce a nd Technol ogy of Fructan s, M. Suzuki and N. Chatterton, eds CRC Press Inc., Boca Raton, FL, pp. 319-352) . The simple hydrolysis of fructan in individual fructose residues has a tremendous advantage over the current process, technically required for the enzymatic conversion of starch into higher fructose syrup. Using fructan as the initiator material, therefore, significantly reduce production costs.

The commercial potential for fructan is extremely high, however, its use is severely limited mainly due to the high production cost. Fructan used in low-calorie foods is currently produced by fermentation culture. The higher polymers synthesized by bacteria are not currently produced on a commercial scale. Isolation from plants would reduce production costs, but fructan is not found in many crops of importance in agriculture. Traditional crops, adapted to large growing regions, such as oats, wheat and barley accumulate fructan, but only at extremely low levels. Fructan is currently harvested from plants on a scale relatively small commercial and only from simple plant species, Cichorium i-ty- > -? s.

Transgenic crops that accumulate fructan through the expression of fructosyl transferase (FTF) chimeric genes, would have a significant advantage over native fructan storage plants, making use of established breeding programs, resistance to pests and adaptation to a variety of Growth regions throughout the world. Examples of the synthesis of fructan have been reported in transgenic plants containing genes from bacterial species, such as Bacillus, Streptococcus and Erwinia (Caimi et al., (1996) Pl-u-t Physiol, 110: 355-363; Ebskamp et al., (1994) Biotechnol .. 12: 272-275; Robert et al., (1996) Planta 155: 528-536). The synthesis of fructan was demonstrated in these plants that do not store fructan, but the accumulation was frequently very low and in tissues where high fructan levels were reported to have a detrimental effect on the development of the plant.

Several important differences were reported between transgenic plants - expressing chimeric bacterial FTF genes and plants that store native fructan. The The most obvious difference was in the size of the synthesized polymers. Transgenic lines containing bacterial FTF genes accumulate fructan with a GP greater than 5000 (Ebskamp et al., (1994) Biotechnol 12: 272-275; Caimi et al., (1995) Plant Physiol. 110: 355-363) . The polymers synthesized in transgenic plants are, therefore, several times larger than fructans that accumulate in plants such as chicory (Cichorium intibus L.) and artichoke (Helinathus tuberosus L.).

Differences in specificity for donor and acceptor molecules have also been reported for FTFs of plants and bacteria. Bacterial enzymes are known to release significant amounts of fructose to water as an acceptor (invertase activity), while plant enzymes do not have invertase activity (Chamberí, R. and Pet it-Glatron, M. (1993) Inulm and Inulin Containing Crops, A. Fuchs, Elsevier Press, Amsterdam pp. 259-266). Fructose, released from sucrose by invertase activity, can not be used to increase the length of a polymer. Bacterial FTFs, therefore, convert sucrose to fructan less efficiently than plant enzymes.

The two classes of FTFs also differ in their affinity for sucrose, the only substrate. The sucrose-sucrose-fructosyltransferase (SST) of artichoke has one Km for the reported sucrose that is approximately 100 mM (Koops, A. and Jonker, H., (1994) J. Exp. Bo t.45: 1623-1631 ). In contrast, the bacterial enzyme has a much lower Km, approximately 20 mM (Chamberí, R., and Petit-Glatron, M. (1991) Bi or ch., J. 275: 35-41). This difference could have a critical effect on the synthesis of fructan, which results in higher or lower levels of accumulation, depending on the concentration of sucrose in the cell. The fundamental differences between FTF enzymes prevent significant predictions regarding the result of gene expression of plants in transgenic tissue, based on the expression of bacterial FTF genes.

The prediction of whether or not fructan would accumulate in a transgenic line containing the FTF genes derived from plants, could be significantly improved if there was a greater understanding of the metabolic pathway of fructan in plants that store native fructan. The currently accepted model for the synthesis of fructan in plants suggests that the synthesis is a reaction in two stages. The initial reaction involves the enzyme sucrose-sucrose-1 transferase (SST). The SST catalyzes the synthesis of a trisaccharide of two sucrose residues. The second stage, chain elongation, is carried out by the enzyme fructan-fructan-fructosyltransferase (FFT), (Edelman J., and Jefford T. (1968) Ne w Phyt ol. 67: 517-531. has applied to all plants that store fructan (ca 45000 species), however, it is largely based on the results of a simple species, Hel ian th us t eros ers us, and has undergone several revisions. SST can only act in the production of long-chain fructan (Van der Ende, W. and Van Laere, A., (1996) J. Exp. Bo 47: 1797-1803). Additional features in the model are necessary and suggest that there is only a rudimentary knowledge of the synthesis of fructan in plants.

Examples of the synthesis of fructan have been reported in transgenic plants containing plant-derived or microbial FTF genes (Vijn, et al., (1997) Th e Pl ant J. 11: 387-398; Smeekens et al., WO 96 / 01904; Van Tunen et al., WO 96/21023; Sevenier et al., (1998) Na tu re Bi ot echn olgy 16: 843-846). This previous work involves the expression of SST genes derived from plants or microbials only in dicotyledonous plants (dicots) transgenic. The present invention describes a method to increase the level of fructan synthesis in transgenic monocotyledonous plants containing SST genes derived from plants or genes ST and FFT derived from plants.

There are numerous differences between monocotyledonous and dicotyledonous plants that inhibit the extrapolation used of cases that occur in one plant based on the results of another. These differences include, but are not limited to, competition for sucrose as a source of energy between biosynthetic pathways in various plant organs and between biosynthesis pathways in different plant species.

Dicots and monocots are known to differ significantly in carbohydrate transport and metabolism. For example, the pea (Pi s um sa t i vum L.) a dicotyledonous, transports glucose-6-phosphate in amyloplasts, the site where the starch is synthesized and stored. In monocots, such as corn, ADPglucose is transported in the amyloplast (Denyer et al., (1966) plant Phys. 2: 779-785). This seemingly simple difference illustrates a profound difference in the metabolic pathways necessary to process various forms of carbohydrates transported in the amyl-plast in the two separate floors.

In sucrose transport in plants also differs between plant species. Specialized cells (basal endosperm transfer cells or BET cells) are adapted for the transport and metabolism of sucrose in corn grains. The majority (greater than 90%) of sucrose transported to maize seeds is believed to be hydrolyzed in the specialized BET layer (Shannon, J. (1972) Pl ant Physol., 45: 198-202. The resulting proteins are transported to the cells of the developed endosperm and re-synthesized as sucrose before entering the starch biosynthesis path In contrast to the maize, the sucrose is transported directly to the tubers of potato plants and enters the path starch without hydrolysis (Oparka, K. and Wright, K. (1998) Plant 1 74: 123-126).

Although poorly understood, exploitation of the differences between monocots and dicots could not be considered a new concept. These differences are what drives the commercialization of herbicides, such as 2,4-D, which is tremendously toxic to dicots, but has no effect on monocot species. In this sense, it seems clear that the recent examples of transgenic dicotyledonous species containing a FTF gene derived from plant (Vijn, et al., (1997) The Pl ant J. 11: 387-398; Smeekens et al., WO 96/01904; Van Tunen et al., WO 96/21023; Sevenier et al., (1998) Wature Biotech chn olgy 16: 843-846), can not support the prediction of successful expression of FTF genes in monocotyledonous species. Variations in carbohydrate concentration, transport and metabolism among plant species, especially between monocots and dicots, are widely too large to allow generalization.

BRIEF DESCRIPTION OF THE INVENTION This invention describes a method for producing fructose polymers of various lengths by means of expression of FTF genes derived from plants in a transgenic monocot and ledon species. More specifically, the invention describes a chimeric gene comprising a tissue-specific promoter, operably linked to the coding sequence for a sucrose-sucrose-fructosyltransferase gene (SST; EC 2.4.1.99), so that the chimeric gene is capable of of transforming a monocot monocot plant cell that results in the production of fructan without harmful effect on the cell of the plant.

The invention further discloses a chimeric gene comprising a tissue-specific promoter, operably linked to the coding sequence for a fruct gene ano-fruct ano-fructosyltransferase (FFT; EC 2.4.1.100), so that the chimeric gene is capable of transforming a transformed plant cell (protecting a chimeric gene comprising a tissue-specific promoter, operably linked to the coding sequence for a sucrose-sucrose-p-ribosyltransferase (SST; EC 2.4.1.99) gene which results in the production of fructan, without harmful effect on the cell of the plant.

The invention also includes a monocotyledonous plant transformed with one or both of the chimeric genes described above, so that the plant produces fructan. The invention also relates to a method for producing fructose or fructose polymers, comprising the growth of the plant, harvesting the plant and extracting fructan from the harvested plant.

The invention also describes a chimeric gene that comprises a tissue-specific promoter, operably linked to the coding sequence for a sucrose-sucrose-p-ribosyltransferase gene (SST; EC 2.4.1.99), so that the chimeric gene is capable of transforming a monocot plant cell that is results in the production of fructose polymers containing 2 to 3 fructose residues, with no detrimental effect on the transformed plant cell.

The present invention is not naturally limited to naturally occurring fructosyl rans ferases, but could equally well be transformed using the modified versions thereof. The modifications could influence the activity of the fructosyl rans ferase, in such a way that, for example, the degree of polymerization or the structure of the fructan produced is altered. In addition, according to the present invention, a simple iltransferase fungal gene or a combination of plant-derived ructosyltransferase genes could be used.

The induced accumulation of fructans in transgenic plants using the principles described herein, will allow the extraction of fructans from these plants for the purpose of fructan production.

Fructans can accumulate in these plants (e.g., in harvested organs, such as roots, leaves, stems and seeds). In addition, the present invention relates to seeds, cuttings or other parts of transgenic plants that are useful for the continuous production of additional generations of these plants.

The fructans produced using transgenic plants of the present invention could be used in various food and non-food applications. Examples include, but are not limited to, human and animal food products, in the production of fructose syrups, in the production of chemicals and plastics, either as such or in a modified form.

The plants of genetically modified crops that incorporate the constructions that code the f r u c t s 111 r a n s s a re mentioned above, will allow the efficient production of polymers of high quality carbohydrates used by man.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a diagram of the cassettes used to express the SST and artichoke FFT genes (SST of 10 kD and 10 kD FFT, respectively) in the transgenic corn endosperm. Each construct also contains the tissue-specific 10 kD zein promoter and the 3 'transcription termination region.

Figure 2 shows the Southern Blot analysis of the leaf tissue of fourteen independently transformed lines that only contain the 10 kD SST cassette. The genomic DNA was digested with the Bgl II restriction enzyme. The complete 2.0 Kb coding sequence of the SST gene was labeled with 32P and used to probe the genomic DNA that was previously transferred to nylon membranes. Copies of multiple intact (indicated by the arrow) and rearranged copies of the SST gene showed that several transgenic maize lines are present.

Figure 3 shows the TLC analysis of the individual seeds of the three transgenic lines containing the intact copies of the 10 kD SST expression cassette. The polymers of Fructose, F; Sucrose, S; and fructan containing 1 or 2 additional fructose residues (DP3 and DP4, respectively). A marker lane (M) containing fructose, sucrose, fructans DP3 and DP4 is also indicated.

Figure 4 shows the Southern Blot analysis of the leaf tissue of twenty independently transformed lines containing the 10 kD FFT and 10 kD SST cassettes. The DNA of the previously shown lines containing at least one intact copy of the SST cassette of kD, was digested with the restriction enzymes Eco Rl and Bam Hl. The complete coding sequence of the FFT gene was labeled with 32 P and used to probe the genomic DNA again. Copies of multiple intact (indicated by the arrow) and rearranged copies of the FFT gene were shown to be present in several transformed lines.

Figure 5 shows the TLC analysis of the individual seeds of two transgenic lines containing intact copies of the 10 kD FFT and 10 kD SST expression cassettes. The fructose polymers, greater than DP3 were demonstrated in the seeds of each of the two lines. The polymers of Fructose F are indicated; Sucrose S and fructan containing 1 and 2 additional fructose residues (DP3 and DP4, respectively). A marker lane (M) containing fructose, sucrose, fructans DP3 and DP4 is indicated.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS In the context of the description, a number of terms will be used. As used herein, the term "nucleic acid" refers to a long molecule that may be single stranded or double stranded, composed of monomers (nucleotides) containing a sugar, phosphate and a purine or pyrimidine. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is involved in the transfer of DNA information in proteins. A "genome" is the complete body of the genetic material contained in each cell of an organism. The term "nucleotide sequence" refers to a DNA or RNA polymer that can be single or double stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.

As used herein, "substantially similar" refers to DNA sequences that could involve base changes that do not cause a change in the encoded amino acid, or that involve base changes that could alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. Therefore, it is understood that the invention encompasses more than the specific exemplary sequences. Sequence modifications are also encompassed, such as deletions, insertions or substitutions in the sequence that produce insignificant changes that do not substantially affect the functional properties of the resulting protein molecule. For example, alteration in the sequence of the gene that reflects the degeneracy of the genetic code, or that results in the production of a chemically equivalent amino acid at a given site is contemplated; in this manner, a codon for the amino acid alanma, a hydrophobic amino acid, could be replaced by a codon encoding another hydrophobic amino acid residue such as glycine, valine, leucine, or Ioleucma. Similarly, changes that result in the substitution of a negatively charged residue for another, such as aspartic acid for glutamic acid, or a positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product. The changes of nucleotides that result in the alteration of the N terminal and C terminal portions of the protein molecule, would not be expected to alter the activity of the protein. In some cases, it may indeed be desirable to make mutants of the sequence to study the effect of the alteration in the biological activity of the protein. Each of the proposed modifications is within the routine of the expert in the art, as is the determination of the retention of the biological activity of the encoded products. Furthermore, the person skilled in the art recognizes that the "substantially similar" sequences encompassed by this invention can also be defined by their ability to hybridize, under severe conditions (0.1X SSC, 0.1% SDS, 65 ° C), with the sequences exemplified here.

"Gene" refers to a fragment of nucleic acid that encodes all or a portion of a specific protein, and includes the regulatory sequences that precede (5 'non-coding) and that follow (3' non-coding) the coding region. "Native gene" refers to the gene as it is found in nature with its own regulatory sequences. "Chimeric gene" refers to a gene comprising the heterogeneous and coding regulatory sequences. "Endogenous gene" refers to the native gene normally found in its natural location in the genome. A "foreign gene" refers to a gene not normally found in the host organism, but which is introduced by the transfer of the gene. The "foreign gene" may also refer to a gene that is normally found in the host organism, but which is re-introduced at a location in the genome where it is not normally found, resulting in one or more additional copies of the sequence of coding of an endogenous gene.

"Coding sequence" refers to a DNA sequence that encodes a specific protein and excludes non-coding sequences.

"Initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides in a coding sequence that specifies the initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Open reading frame" refers to the amino acid sequence encoded between the initiation of translation and the termination codons of a coding sequence.

"RNA transcription" refers to the product that results from the catalyzed transcription of RNA polymerase from a DNA sequence. When RNA transcription is a perfect complementary copy of the DNA sequence, it is referred to as the "primary transcript" or it could be an RNA sequence derived from the post-transcriptional processing of the primary transcript. "Messenger RNA" (mRNA) refers to the RNA that can be translated into the protein by the cell. "CDNA" refers to a double-stranded DNA, a strand of which is complementary and derived from the mRNA by reverse transcription.

As used herein, appropriate "regulatory sequences" refer to nucleotide sequences located upstream (5 '), in, and / or downstream (3') to a coding sequence, which controls transcription and / or expression of the coding sequences. These regulatory sequences include promoters, leader translational sequences, transcription termination sequences and polyadenylation sequence. In artificial DNA constructs, regulatory sequences can also control the transcription and stability of anti-sense RNA.

"Promoter" refers to a DNA sequence is a gene, usually upstream (5 ') to its coding sequence, which controls the expression of the coding sequence providing recognition for the RNA polymerase and other factors required for the proper transcription. A promoter could also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of the initiation of transcription in response to physiological or developmental conditions. It could also contain improvement elements.

An "enhancer" is a DNA sequence that can stimulate the activity of the promoter. It could be an innate element of the promoter or a heterologous element introduced to improve the level and / or tissue specificity of a promoter. "Constitutive" promoters refer to those that direct the expression of the gene in all tissues and show little temporal or developmental regulation. The "tissue specific" or "developmental specific" promoters as referred to herein, are those that direct gene expression almost exclusively in specific tissues, such as leaves and seeds, or in specific stages of development in a tissue, such as early or late embryogenesis, respectively.

The term "operably linked" refers to nucleic acid sequences in a single nucleic acid molecule, which are associated so that the function of one is affected by the other. For example, a promoter is operably linked to a structural gene (i.e., a gene encoding a fructosyltransferase) when it is capable of affecting the expression of such a structural gene (i.e., the structural gene is under the transc iptional control of the promoter).

The term "expression", as used herein, is meant to mean the production of a functional terminal product encoded by a gene. More particularly, "expression" refers to sense transcription (mRNA) or antisense RNA derived from the fragments of the nucleic acid of the invention which, in conjunction with the cell's protein apparatus, results in altered levels of the product. proteinaceous. "Altered levels" refers to the production of gene products in transgenic organisms in amounts or proportions that differ from normal or non-transformed organisms.

"Non-coding 3 'sequences" refers to the portion of the DNA sequence of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of acid traits polyadenylic to the 3 'end of the mRNA precursor.

"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the trans formed nucleic acid fragments are referred to as "transgenic" organisms.

"Fructosyltransferase" refers to a protein encoded by any of several plant genes that have the property of producing a carbohydrate polymer, consisting of repeated fructose residues. The repeated fructose residues could be linked by the < x2-l or a link < x2-6 or any combination of the two types of links. The polymer of the repeated fructose residues could contain a terminal glucose residue, derived from a sucrose molecule, and at least two fructose residues. The polymer of the fructose residues repeated in any form, with any combination of bonds, and with any number of fructose residues, is generally referred to as "fructan".

A "fructosyltransferase gene" or "FTF" refers to to the DNA sequence encoding a fructosyltransferase protein. The term "deleterious effect" as used herein, refers to a direct or indirect harmful effect on a plant or plant cell as a result of the accumulation of fructan, so that the plant or plant cell is prevented from performing certain functions which include, but are not limited to, synthesis and transport of carbohydrates within a cell and through the plant, regeneration of plants or transgenic tissue, development of the plant or plant cell at maturity, or the ability to pass the trait or desired traits to the progeny.

The present invention describes chimeric genes comprising tissue-specific regulatory sequences, FTF coding sequences and a transcription termination region. The chimeric gene is capable of regulating the synthesis of a fructose polymer using sucrose as a substrate when it is expressed in a transgenic monocot plant, wherein the expression of the gene FTF results in the synthesis of new fructose polymers, used in numerous food and industrial applications. A transgenic maize plant (Zea mays) that properly expresses the FTF gene, is distinguished from a generic plant of the same species by the presence of the accumulation of fructan in the mature seeds.

The transfer of the nucleic acid fragments of this invention in a plant, directs the expression of the protein in a manner that results in the accumulation of this used polymer, without referring to the loss or alteration of the polymer due to the degrading enzymes of the plant during harvest, transport or storage and without the loss of the established by-products of any particular species. Transgenic crops containing chimeric genes comprising tissue-specific regulatory sequences, the FTF gene and a transcription termination region, will provide a renewable source of small molecular weight (GP 2-3) and large fructose polymers. The accumulation of fructan will be determined in part, by the level of expression of the chimeric gene in the transformed cultures. The level of expression depends, in part, on tissue-specific expression signals, the number of copies of the gene integrated into the plant genome and the location of the gene integration; the accumulation of fructan could also be subject to substrate availability. The amount of substrate available to the enzyme depends on the species (including mutants within a species), the type of tissue where the expression, the subcellular localization of the expression and the stage of development of a particular plant. The stability of the introduced protein could also influence the accumulation of fructan and depends in part on its proper processing, intracellular targeting and its ability to function in a foreign environment.

The successful expression of a gene with metabolic properties of carbohydrates such as the SST and FFT genes in a transgenic plant would require consideration of the following factors: (1) the transformed species, (2) the specific tissue in which the expression is going to present , (3) and the time of expression. All these factors must be carefully coordinated for the production of fructan to be present in a transgenic cell, without harmful effect.

Expression of a gene with sucrose methylating activity, such as an FTF protein, in a transgenic plant species would not necessarily create the same, or even a desired effect when expressed in a different plant species. Differences in carbohydrate profiles between species suggest that a specific enzyme for sucrose will not always have enough substrate available to produce the same result, when it is expressed in several species. It is well established that the availability of sucrose as a substrate not only varies widely from species to species, but also in mutants within the same species, (Lampe et al., (1931) Bo t. Ga z., 51: 337 -380).

The mechanisms for sucrose transport and tissue accumulation also vary widely from one species to another. Sucrose hydrolysis is an integral part of the mechanism of import into developed maize seed, (Porter et al., (1985) Pl ant Phys., 77: 524-531), but it is not a prerequisite for transport to develop the soybean embryo (Thorne, (1982) Pl ant Phys., 70: 953-958), or the wheat endosperm (Jenner, Aust. J. Pl ant Phys., 1: 319-329) (1974)). Therefore, the expression of an FTF in the seed of a species, could have access to an abundance of sucrose, however, the synthesis of fructan in the seed of another species could be made severely by the accumulation of hexose sugars instead of sucrose.

Tissue specific expression and development of a gene could be intrinsic to the promoter, the 3 'non-coding region or combinations of the two, used in chimeric constructions. The promoters used to activating the expression of the gene in transgenic plants can be derived from many sources provided that the promoter (s) chosen have sufficient transcriptional activity to perform the invention by expressing the mRNA in the desired host tissue. Preferred promoters are those that allow expression specifically in seeds. Examples of seed-specific promoters include, but are not limited to, the promoters of seed storage proteins. Seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly stage-specific and organ-specific manner (Higgins et al. (1984) Ann. Rev. Pl ant. Physi ol. 35: 191-221 Goldberg et al. (1989) Cell 56: 149-160; Thompson et al. (1989) BíOEs says 10: 108-113). In addition, different seed storage proteins could be expressed at different stages of seed development.

Numerous examples currently exist for seed-specific expression of seed storage protein genes in transgenic plants. These genes include monocot genes, such as, for example, barley ß-hordein (Marris et al., 1988).

Plant Mol. Biol. 10: 359-366) and wheat glutein (Colot et al (1987) EMBO J. 6: 3559-3564). In addition, promoters of seed-specific genes, operably linked to heterologous coding sequences in chimeric gene constructs, also maintain their spatial and temporal expression pattern in transgenic plants. Such examples include the binding of either the 2S albumin promoters of phaseolin or the sequence encoding the 2S albumin of Brazil nut and expressing such combinations in tobacco, Arabidopsis or Brassica nopus (Altenbach et al (1989) Plant Mol. Biol. 13: 513-522; Altenbach et al. (1992) Plant Mol. Biol. Id: 235-245; De Clercq et al. (1990) Plant Physiol. 54: 970-979), bean lectin and bean β-phaseolin promoters to express luciferase (Riggs et al (1989) Plant Sci. 63: 47-57), and wheat glutein promoters to express chloramphenicol acetyl transferase (Colot et al (1987) EMBO J. 6: 3559-3564.

Of particular use in the expression of the nucleic acid fragments of the invention, will be the promoters of several extensively characterized corn seed storage protein genes, such as endosperm-specific promoters of the 10 kD zein gene (Kirihara et al. al. (1988) Gene 71: 359-370), the zein gene of 15 kD (Hoffman et al. (1987) EMBO J. 6: 3213-3221; Schernthaner et al. (1988) EMBO J. 7: 1249-1253; Williamson et al. (1988) Plant Physiol. 88: 1002- 1007), the 27 kD zein gene (Prat et al (1987) Gene 52: 51-49; Gallardo et al (1988) Plant Sci. 54: 211-281) and the 19 kD zein gene ( Marks et al (1985) J. Biol. Chem. 260: 16451-16459). The relative transcriptional activities of these promoters in maize have been reported (Kodrzyck et al. (1989) Plant Cell 1: 105-114) providing a basis for choosing a promoter for use in the constructions of chimeric genes for maize. In addition, promoters that direct the expression of genes encoding enzymes involved in starch biosynthesis, could be used in the practice of this invention. These include the 5 'regulatory sequences of the sucrose synthase (Yang, NS and Russell, D. (1990) Proc. Nati, Acad. Sci. 87:41 -4188) and the granulation or starch binding synthase I genes. wax (Unger et al. (1991) Plant Physiol. 96: 124).

The promoter elements could be derived from other genes of starch synthase (soluble isoforms and granule binding) when they become available, and from the sh2 genes (Bhave et al. (1990) Plant Cell 2: 581-588) and bt2 ( Bae et al. (1990) Maydica 35: 317-322) whose products they constitute the enzyme ADP-glucose pyrophosphorylase. It is envisioned that the introduction of enhancers or elements similar to enhancers in other promoter constructions will also provide increased levels of primary transcription to carry out the invention. These would include viral enhancers such as that found in the 35S promoter (Odell et al. (1988) Pl ant Mol. Biol.10: 263-272), opine gene enhancers (Fromm et al. (1989) Pl ant Cel 1: 977-984), or enhancers of any other source that results in increased transcription when placed in a promoter operably linked to the nucleic acid fragment of the invention.

The isolated bystanders of corn genes Adh-1 and Bz-1 (Callis et al (1987) Gen is Dev. 1: 1183-1200), and intron 1 and exon 1 of the corn Shrunken-1 gene (sh- 1) (Maas et al (1991) Pl ant Mol. Bi ol. 16: 199-207) could also be used to increase the expression of introduced genes. The results with the first intron of the maize alcohol dehydrogenase gene (Adh-1), indicate that when this element of DNA is placed inside the transcriptional unit of a heterologous gene, mRNA levels can be increased by 6, 7 times the normal levels. Similar levels of intron enhancement have been observed using intron 3 of a corn actin gene (Luehrsen, K.R. and Walbot, V. (1991) Mol. Gen. Gen e t.225: 81-93). The improvement of gene expression by Adhl intron 6 has also been observed (Oard et al (1989) Plant Cel l Rep 8: 156-160). Exon 1 and intron 1 of the maize sh-1 gene have been shown to individually increase the expression of reporter genes in corn suspension cultures by 10 and 100 fold, respectively. When used in combination, these elements have been shown to produce up to 1000-fold stimulation of reporter gene expression (Maas et al. (1991) Pl an t Mol. Biol. 1 6: 199-207).

Any 3 'non-coding region capable of providing a polyadenylation signal and other regulatory sequences that might be required for appropriate expression may be used to carry out the invention. This would include the 3 'end of any storage protein such as the 3' end of the 10 kd, 15 kd, 27 kd and alpha zein genes, the 3 'end of the bean phaseolin gene, the 3' end of the gene β-soybean conglicin, the 3 'end of the viral genes such as the 3' end of the 35S and 19S cauliflower mosaic virus transcripts, the 3 'end of the opine synthesis genes, the 3' ends of 1,5- ribulose carboxylase bisphosphate or chlorophyll binding protein a / b the 3 'end sequences of any source, so that the sequence employed provides the necessary regulatory information within its nucleic acid sequence, to result in appropriate expression of the combination of the promo/ coding region to which it is linked operably. There are numerous examples in the art that show the usefulness of the different 3 'non-coding regions (for example, see Ingelbrecht et al. (1989) Plant Cell 1: 671-680).

A number of genes from plant sources encoding enzymes with FTF activity have been isolated and sequenced. These include the SST and FFT genes of onion (Allium layer L.), barley (-or euis vulgare L.) and artichoke (Helianthus tuberosus); (Vijn et al., (1997) Plant J. 11: 387-398; Sprenger et al., (1997) Febs Lett. 400: 355-358; Van Tunen et al., WO 96/21023; Smeekens et al. , WO 96/01904). Among the preferred ones are the SST- and FFT genes derived from artichoke plants.

The? ST and FFT genes can be isolated by the techniques routinely employed by the person skilled in the art for the isolation of genes, when the nucleotide sequence of the desired gene, or when the sequence of a homologous gene of another organism is known. The sequence information near the desired gene can be used to prepare the oligonucleotide probes for identification and isolation of the entire gene from an appropriate genetic library. This library could be a genomic library, where the coding region could be contained in a single DNA fragment or it could be contained in several different DNA fragments. Alatively, the library could be a cDNA library, where the probability of isolation of a cDNA clone comprising the entire coding region as a contiguous sequence is grea In any case, the appropriate clones can be identified by DNA-DNA hybridization with probes corresponding to one or more portions of the desired genes. Alatively, the oligonucleotide primers can be prepared and used as PCR primers to subsequently amplify and isolate all or part of the coding region of the genomic DNA, or of the genomic or cDNA libraries described above.

Several different tests can be used to detect the expression of the chimeric genes in seeds of the transformed plants. RNA transcripts, specific to the FTF genes, could be detected by Southern or northern analysis. The FTF protein can be extracted, detected and quantified immunologically by methods known to those skilled in the art. Alatively, the seed tissue could be crushed and extracted with a polar solution, the isolation and concentration of polysaccharides, including fructans, which can then be detected by: TLC analysis, combined with a specific cestose stain (Wise et al., ( 1995) Anal, Chem. 27: 33-36); HPLC analysis using fructan standards (Chaton et al. (1993) In: Fuchs A. ed. Inulin and Inulin-containing crops, Elsevier, Amsam pp. 93-99); or continuous hydrolysis and an enzymatic binding test (Brown, C. and Huber, S. (1987) Physiol. Plant 70: 537-543).

Various methods for introducing a DNA sequence (chimeric constructs containing SST or SST / FFT genes) into eukaryotic cells (ie, transformation) of higher plants, are available to those skilled in the art (see EPO publications 0 295 959 A2 and 0). 138 341 Al). Such methods include high speed ballistic bombardment with metal particles coated with the nucleic acid constructs (see Klein et al (1987) Nature (London) 327: 70-73, and see U.S. Pat. 4,945,050), as well as those based on transformation vectors based on Ti and Ri plasmids of Agrobacum ssp. , particularly the binary type of these vectors. Vectors derived from Ti transform a wide variety of higher plants, including monocot and dicotyledonous plants, such as soybeans, cotton and rapeseed (Pacciotti et al. (1985) Bio / Technology 3: 241; Byrne et al. (1987) Plant Cell, Tissue and Organ Culture 8: 3; Sukhapinda et al. (Plant Mol. Biol. 8: 209-216; Lorz et al. (1985) Mol. Gen. Genet. 155: 178-182; Potrykus et al. 1985) Mol. Gen. Genet, 155: 183-188).

Other methods of transformation for chimeric constructions containing SST or SST / FFT genes are available to those skilled in the art, such as direct taking of foreign DNA constructs (see EPO publication 0 295 959 A2), and electroporation techniques (See Fromm et al. (1986) Nature (London) 3: 791-793). Once they are transformed, the cells can be regenerated by those skilled in the art. Also relevant are several methods recently described for introducing nucleic acid fragments into commercially important crops, such as rape seed (see De Block et al. (1989) Plant Physiol. 51: 694-701), sunflower (Everett et al. , (1987) Bio / Technology 5: 1201-1204), soy (McCabe et al (1988) Bio / Technology 6: 923-926; Hinchee et al (1988) Bio / Technology 6: 915-922; Chee et al. (1989) Plant Physiol. 51: 1212-1218; Christou et al. (1989) Proc. Nati, Acad. Sci USA 86: 7500-7504; EPO Publication 0 301 749 A2), and corn (Gordon-Kamm et al. (1990) Plant Cell 2 603-618; Fromm et al. (1990) Bio / Technology 8: 833-839). One skilled in the art is familiar with other means for the production of transgenic maize plants that include the introduction of DNA into the protoplasts and the regeneration of plants from these protoplasts (Omirulleh et al. (1993) Plant Mol. Biol. 21: 415-423), electroporation of intact tissues (D'Hulluin et al. (1992) Plant Cell 4: 1495-1505; Laursen et al. (1994) Plant Mol. Biol. 24: 51-61), regulated silica carbide by the transformation of corn cell fiber (Kaeppler et al (1992) Theor, Appl. Genet, 84: 560-566; Frame et al. (1994) Plant J. 6: 941-948). In addition to the method of particle bombardment of corn callus cells described above, one skilled in the art is familiar with the bombardment of particles of suspension cultures or of corn shield to produce fertile transgenic plants (Koziel et al., 1993). ) Bio / Technology 11: 194 -200; Walters et al. (1992) Plant Mol. Biol. 18: 189-200).

Once the transgenic plants are obtained by one of the methods described above, it will be necessary to screen the individual transgenics for those that effectively exhibit the desired phenotype. It is well known to those skilled in the art that individual transgenic plants carrying the same construction could differ in expression levels; This phenomenon is commonly referred to as "position effect". For example, when the construction is question is designed to express higher levels of the gene of interest, the individual plants will vary in the amount of the protein produced and in this way, in the enzymatic activity; this in turn will affect the phenotype. This should not be seen as a limitation of the present invention, but as a practical matter that is appreciated and anticipated by the person skilled in the art. Therefore, the art expert will develop methods to screen large numbers of transformations. The nature of these screenings in general will be chosen in practical matters, and is not an inherent part of the invention.

EXAMPLES The present invention is further defined in the following examples. It will be understood that the examples are given for illustration only and the present invention is not limited to the uses described in the examples. The present invention can be used to generate transgenic maize plants whose seed carbohydrate profile is altered by the accumulation of fructose polymers, and where its properties are useful, such as but not limited to, food, paper, plastics, adhesives or painting. From the above discussion and the following examples, an expert in the art can find out and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. All modifications are intended to fall within the scope of the rei indications made.

EXAMPLE 1 Chimeric Construction for the Expression of the SST Gene of Artichoke in Zea ma vs L. Transgenic A construct designed for tissue-specific expression of the artichoke SST gene in the maize endosperm was assembled by replacing the 35S promoter of the Cauliflower Mosaic Virus (CaMV) in the plasmid pSST403 (Van Tunen et al., WO 96/21023 ) with 10 kD zein specific for the maize endosperm, promoter of the seed storage gene (Kirihara et al. (1988) Gen e 71: 359-370). The complete SST coding sequence contained in p? ST403 (Van Tunen et al., WO 96/21023) was isolated by digesting with the restriction endonuclease enzymes Ncol and HindIII. The isolated sequence was added to the plasmid pCyt-SacB (Caimi et al (1996) Pl a nt Physi o 1. 110: 355-363) which contains a 10 kD zein promoter and the 3 'termination region. The corn endosperm expression cassette, containing the 10 kd promoter, the SST coding sequence (including the native vacuole target and secretory signals) and the 3 'end of 10 kD, designated SST 10 kD (Figure 1 ), was isolated by digesting with Smal and Sali, then ligated into the plasmid KS17. The KS17 vector contains a hygromycin resistance gene (HPT) using as the selected marker. The final vector was designated SST-17 of 10 kD, it was used directly for transformation into corn by particle bombardment.

Material and Transformation of the Plant The expression vector of the 10 kD SST-17 plant and a plasmid vector encoding a selected marker (pDetric) were co-t transformed into the embryogenic corn callus derived from crosses of the cultivated lines A188 and B73 (Armstrong et al. al. (1991) Maize Gen e t i cs Coopera t i on Newsl e t t er 65: 92-93) by microprojectile bombardment (Klein et al., (1987) Wature 327: 70-73). The transformed embryogenic cells were recovered in the medium containing either glufosinate-ammonium or chlor sulfuron. The selected pDetric marker contains the BAR gene (Thompson et al. (1987) The EMBO Jo urn 6: 2519-2523), which encodes phosphinotricin acet iltransphrase, under the control of the 35S promoter. pALSLUC (Fromm et al (1990) Bi o techn olgy 8: 833-839), a plasmid vector encoding a mutant acetolactate synthase (ALS) gene that confers resistance to chlor sulphon, could also be used as a marker selected. The expression of the mutant ALS gene is regulated by the CaMV 35S promoter. The transgenic shots transferred to 12-inch wells containing METROMIX ™ soil (Scotts-Sierra company) and grew to maturity in the greenhouse. R1 mature seeds of the original transformations were grown in the greenhouse and planted directly in the field.

Analysis of Transaénicas Plants that Express the SST Gene The detection of the SST gene in transgenic plants was carried out by PCR analysis, using oligonucleotide primers specific for the SST gene: SST-1: 5 '-ATGAATCCTTTCATCCACCACGACCACCCCTCTC-3' (SEQ ID NO: 1) SST-2: 5 '-CCCAGGAAGAGGGAAAGGATTGAGTTCTGCTTCCCC-3' (SEQ ID NO: 2) They confirm the presence of the SST gene in transgenic tissue and estimate that the copy number was made by Southern Blot analysis, using the complete 2.0 kb SST coding sequence. Southern analysis demonstrated the presence of multiple intact and rearranged copies of the SST gene in the transgenic lines (Figure 2).

Carbohydrate Analysis of Transgenic Corn Lines Containing the SST Gene Individual seeds of the transgenic lines were harvested at 25-35 days post-pollination (DPP) for the detection of fructose polymers. The seeds were crushed with a mortar and pestle. A small amount of water (200-400 uL) was added and the mixture was heated at 80 ° C for 10 minutes. The homogenized tissue was centrifuged at 10,000 s g for 10 minutes and 2 uL of aqueous solution stained on silica TLC plates HP-K (Whatman Scientific, Clifton, NJ). The plates of the TLC developed twice in butanol: propanol: water (3: 14: 4). Fructan was detected by urea-phosphoric acid staining (Wise et al (1955) Ana I. Chem. 27: 33-36). The analysis showed that the control seeds (not transformed) did not contain fructan. The TLC plates also showed that the seeds expressing the SST gene accumulated fructan with a degree of polymerization (GP) of 3 (Figure 3).

EXAMPLE 2 Chimeric Construction for the Expression of the FFT Artichoke Gene in Zea ma vs L. Transaénico A construct designed for tissue-specific expression of the artichoke FFT gene in the maize endosperm was assembled by replacing the promoter 35S Cauliflower Mosaic Virus (CaMV) in the plasmid PSST403 (Van Tunen et al., WO 96/21023) with corn endosperm-specific 10 kD zein, promoter of the seed storage gene (Kirihara et al. (1988) Gen e 71: 359-370). The complete FFT coding sequence contained in pSST403 (Van Tunen et al., WO 96/21023) was isolated by digesting with the restriction endonuclease enzymes Ncol and BamHI. The isolated sequence was added to the plasmid pCyt-SacB (Caimi et al. (1996) Pl a n t Phys i ol. 110: 355-363) containing a 10 kD zein promoter and the 3 'termination region. Pcyt-SacB was digested to Ncol and BamHI to remove the SacB region. The corn endosperm expression cassette, containing the 10 kd promoter, the FFT coding sequence (including native vacuole target signals and secretory signals) and the 3 'end of 10 kD, designated FFT 10 kD (Figure 1) ), was isolated by digesting with Smal and Sali, then ligated into the plasmid KS17. The final vector was designated 10 kD FFT-17, used directly for transformation into corn by particle co-bombardment with the 10 kD SST-17 plasmid, and pDetric described in Example 1. Transformation, regeneration and growth The mature plants were by the methods described in Example 1.

Analysis of Transaénicas Plants that Express the Genes S? T and FFT The detection of the FFT gene in transgenic plants co-bombarded with the SST and FFT genes was carried out by PCR analysis, using oligonucleotide primers specific for the FFT coding sequence: FFT-1: 5 '-CCCCTGAACCCTTTACAGACCTTGAACATGAACCCCA-3' (? EC ID NO: 3) FFT-2: 5 '-GGGCGGAAATCTTGAGAGATGCTTTCACACTCGTACC-3' (SEQ ID NO: 4) They confirm the presence of the FFT gene in transgenic tissue and estimate that the copy number was made by Southern Blot analysis, using the complete 2.0 kb FFT coding sequence. Southern analysis demonstrated the presence of multiple intact and rearranged copies of the FFT gene in the transgenic lines (Figure 4).

Carbohydrate Analysis of Corn Lines Containing SST and FFT Genes Transgenic seeds expressing the S? T and FFT genes were harvested at 25-35 DPP. Isolation and detection of fructan was described in Example 1. Fructan was detected by urea-phosphoric acid staining (Wise et al (1955) Ana l Ch. 27: 33-36). The analysis showed that the control seeds did not contain fructan. TLC plates also showed that the seeds expressing the SST and FFT genes accumulated fructan with a GP much higher than the lines containing the SST gene alone (Figure 5). The results show that the FFT gene acts as a factor of chain elongation, which synthesizes fructan with a GP of at least 20 (the limit of detection by TLC) in transgenic seeds.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) ADDRESS: E.l. DU PONT DE NEMOURS AND COMPANY (B) STREET: 1007 MARKET STREET (C) CITY: WILMINGTON (D) STATE: DELAWARE (E) COUNTRY: USA (F) POSTAL CODE: 19898 (G) TELEPHONE: 302-992-4929 ( H) TELEFAX: 302-773-0164 (I) TELEX: 6717325 (ii) TITLE OF THE INVENTION: TRANSGENIC CROPS? THAT ACCEPT FRUCTOSE POLYMERS AND METHODS FOR THEIR PRODUCTION (iii) NUMBER OF SEQUENCES: 4 (iv) DESCIFRABLE FORM BY COMPUTER: (A) TYPE OF SUPPORT: Flexible Disk 3.50 INCHES (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: MICROSOFT WINDOWS 95 (D) PROGRAMMING ELEMENTS: MICROSOFT WORD VERSION 7. OA (v) CURRENT PRESENTATION DATE: (A) NUMBER OF APPLICATION: 60 / 077,727 (B) DATE OF SUBMISSION: MARCH 12, 1998 (C) CLASSIFICATION: (vi) INFORMATION FROM THE LAWYER / MANDATORY: (A) NAME: MAJARÍAN, WILLIAM R. (B) REGISTRATION NUMBER: 41,173 (C) NUMBER OF RE FERENC IA / REGÍ STRO: BB-1082-A (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (vii) IMMEDIATE SOURCE: (B) CLON: SST-1 (xi) DESCRIPTION OF THE SEQUENCE:? EC ID NO: 1: ATGAATCCTT TCATCCACCA CGACCACCCC TCTC 34 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (vii) IMMEDIATE SOURCE: (B) CLON: SST-2 . { xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CCCAGGAAGA GGGAAAGGAT TGAGTTCTGC TTCCCC 36 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (vii) IMMEDIATE SOURCE: (B) CLON: FFT-1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCCCTGAACC CTTTACAGAC CTTGAACATG AACCCCA 37 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: other nucleic acid (vii) IMMEDIATE SOURCE: (B) CLON: FFT-2 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: A: GGGCGGAAAT CTTGAGAGAT GCTTTCACAC TCGTACC 37 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (10)

1. A method for increasing the level of fructan that accumulates in the cell of a monocot plant, characterized in that it comprises: a) preparing at least one chimeric gene comprising a plant fructosiltrans ferase gene, operably linked to the appropriate regulatory sequences that function in the monocotyledonous cells; b) transforming a monocot and ledonase cell with at least one chimeric gene; Y c) regenerate a monocot and transgenic led plant of the monocot and transformed cell wherein the level of fructan that accumulates in the cells of the transgenic monocot plant is increased when compared to the level of fructan that accumulates in the cells of a monocot plant comprising cells that do not contain at least one chimeric gene .

2. The method of claim 1, characterized in that the fructosyltransferase gene of the plant encodes a sucrose-sucrose-fructosyltransferase.

3. The method of claim 2, characterized in that the monocot ileum cell is transformed with a second chimeric gene comprising a plant fructose-fructose-fuctosyltransferase gene linked to the appropriate regulatory sequences that function in the monocot monoclinic cells, and in where the fructan that accumulates in the cells of the transgenic monocot banana plant has a degree of polymerization of at least 20.

4. The method of claim 1, characterized in that the appropriate regulatory sequences include a tissue-specific promoter.

5. The method of claim 4, characterized in that the tissue specific promoter directs the expression of the fructosi 1 transferase gene operably linked in the seeds of the transgenic monocot plant.

6. The method of claim 1, characterized because the transgenic monocot monocot plant is Zea mays.

7. A chimeric gene, characterized in that it comprises a plant fructosyl transferase gene operably linked to the appropriate regulatory sequences that function in monocotyledonous cells.

8. The chimeric gene of claim 7, characterized in that the fructosyl transferase gene of the plant is a member selected from the group consisting of a sucrose-sucrose-fructosyl trans ferase gene and a f uctose-fuctose-fuct gene. osi lt ransferase.

9. The transgenic monocot monocot plant, characterized in that it is produced by the method of claim 1.

10. The seeds of the plant of claim 9, characterized in that the seeds comprise the chimeric genes comprising a plant fructosi ltransferase gene operably linked to the appropriate regulatory sequences that function in the monocotyledonous cells.