BRPI0913562B1 - METHOD TO INTENSIFY THE PRODUCTIVITY OF A FRUTAN BIOSYNTHETIC PATHWAY AND GENETIC CONSTRUCTION - Google Patents

Abstract

MODIFICATION OF FRUTAN BIOSYNTHESIS, INCREASING PLANT BIOMASS AND INTENSIFYING THE PRODUCTIVITY OF BIOCHEMICAL PATHWAYS IN A PLANT. The present invention relates to the modification of fructan biosynthesis in plants and, more particularly, to methods of manipulating fructan biosynthesis in photosynthetic cells, and to nucleic acids and related constructs. The present invention also relates to increasing plant biomass and, more particularly, to methods of enhancing biomass yield and/or yield stability, including growth of shoots and/or roots in a plant, and to nucleic acids and related constructs. The present invention also relates to methods of enhancing the productivity of biochemical pathways and, more particularly, to fusion proteins in plants, and to nucleic acids and related constructs.

Description

FIELD OF INVENTION

[001] The present invention relates to the modification of fructan biosynthesis in plants and, more particularly, to methods of manipulating fructan biosynthesis in photosynthetic cells, and to nucleic acids and related constructs.

[002] The present invention also relates to increasing plant biomass and, more particularly, to methods of intensifying biomass yield and/or yield stability, including growth of shoots and/or roots in a plant, and the nucleic acids and related constructs.

[003] The present invention also relates to methods of enhancing the productivity of biochemical pathways and, more particularly, to fusion proteins in plants, and to nucleic acids and related constructs.

BACKGROUND OF THE INVENTION

[004] Fructans are a type of water-soluble carbohydrate, whose primary function is to provide a readily accessible energy reserve for plant growth. Fructans are associated with several advantageous traits in grasses, such as cold tolerance to aridity, increased shoot survival, increased persistence, good new growth after mowing or grazing, improved recovery from stress, premature spring growth, and increased nutritional quality.

[005] The synthesis and metabolism of fructan in grasses and cereals are complex. Fructans consist of linear or branched fructose chains linked to sucrose. The chain length of vegetable fructans varies from three to a few hundred fructose units. Different types of fructans can be distinguished based on the types of bonds present. In perennial ryegrass, three types of fructans have been identified: inulins, neoseries of inulins and neoseries of levans, with four fructosyl transferase (FT) enzymes involved in this fructan profile (Figure 6). The enzyme 1-SST (sucrose: sucrose 1-fructosyl-transferase) catalyzes the first step in fructan biosynthesis, while the remaining enzymes elongate the growing fructose chain (1-FFT: fructan: fructan 1-fructosyl-transferase, 6G-FFT: 6-glucose fructosyl-transferase, and 6-SFT: sucrose: ftutose 6-fructosyl-transferase). The enzymes 1-FEH or 6-FEH (phthutoexohydrolase) reduce the length of the fructan chain by releasing fructose molecules.

[006] Fructans represent the main non-structural carbohydrate in 15% of plant species and play a key role in forage quality. Grazing ruminant cattle on high-fructan diets exhibit improved animal performance.

[007] In grasses, the level and composition of fructans in stems and leaf sheaths have been increased through the engineered expression of fructosyltransferase (FT) genes.

[008] However, manipulating biochemical pathways by manipulating the activity of enzymes in the pathways can be difficult, because of the pathways in which the various enzymes and their substrates can interact.

[009] Therefore, it would be desirable to have improved manipulation methods for manipulating biochemical pathways, particularly in plants. For example, it would be desirable to have methods of manipulating fructan biosynthesis in plants, including grass species such as LoHum and Fescue, and cereals such as wheat and corn, whereby the production of e.g. pasture grasses with improved grass quality, leading to improved pasture production, improved animal production and reduced environmental pollution, bioenergy grasses with enhanced biomass yield, e.g. for bioethanol production, and, e.g., cereals with enhanced biomass yield, e.g. grains and biomass increased.

[010] Nucleic acid sequences, which encode some of the enzymes involved in the fructan biosynthetic pathway, have been isolated for certain plant species. For example, PCT/AUO1/00705 to the present applicants describes fructosyl transferase homologues from LoHum and Festuca. However, there remains a demand for materials useful in modifying fructan biosynthesis in plants, and also for engineering fructan accumulation in different parts of the plant.

[011] It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

SUMMARY OF THE INVENTION

[012] Applicants have found that it is possible to nutritionally enhance plants, for example, pasture grasses, and/or increase plant biomass by spatially reprogramming the fructan biosynthesis pathway in photosynthetic cells. Using this process, it is possible to compel the accumulation of fructan in leaf blades, the plant organs that are primarily grazed by livestock, and which normally may not accumulate fructan. Therefore, the accumulation of fructans, especially those that exhibit a high degree of polymerization (high GP fructans), provides more accessible nutrition for grazing animals. Fructans accumulate in stems and leaf sheaths, with leaf fructans forming only during periods when CO2 assimilation outperforms growth. Pasture grasses can be nutritionally enhanced by expression of fructan genes in photosynthetic cells, where sucrose is synthesized, thus compelling the accumulation of fructan preferentially in leaf blades and providing more energy to grazing livestock.

[013] Fructans in pasture grasses contribute significantly to the energy readily available in feed for grazing ruminant animals. Fermentation processes in the rumen require considerable readily available energy. Improving the energy readily available in the rumen can increase the efficiency of digestion in the rumen. An increased efficiency of digestion in the rumen leads to an improved conversion of pasture protein fed to the ruminant animal into milk or meat and a reduction in nitrogen expenditure.

[014] Applicants have also found that reprogramming photosynthetic cells for extended life, for example, by delaying leaf senescence, helps to increase plant biomass.

[015] Applicants have also found that it is possible to enhance the productivity of a biochemical pathway by coordinating enzymatic activity in the pathway, through a genetic construct that encodes a fusion, more preferably a translational fusion, of two or more enzymes from of the road.

[016] Although the applicant does not wish to be restricted by theory, it is envisaged that by bringing two enzymes in a pathway into close proximity, for example, by expression of a translational fusion, the expression of the individual enzymes can be coordinated, e.g. This improves the efficiency of the road. For example, by expressing a translational fusion of two or more FT genes (e.g., Lpl-SST and Lp6G-FFT), problems associated with differences in the expression patterns of these genes, independently integrated into the plant genome, can be relieved, resulting in the conversion of sucrose molecules directly into fructans, those exhibiting a low degree of polymerization (low GP fructans) and a high degree of polymerization (high GP fructans). Furthermore, TF proteins can physically associate with each other to form a metabolic channel for the efficient biosynthesis of fructans.

[017] Furthermore, the expression of TF genes in photosynthetic cells, leading to the accumulation of low and high GP fructans in photosynthetic cells, can lead to a release from photosynthesis inhibition mechanisms, facilitating the capture of solar energy and increased CO2 fixation.

[018] Therefore, applicants have found that the reprogramming of photosynthetic cells for extended life and enhanced fructan biosynthesis facilitate the capture of solar energy and increases the production of plant biomass, including the growth of shoots and/or roots.

[019] Furthermore, since the accumulation of low and high GP fructans has been associated with plant tolerance to abiotic stress, such as cold and aridity; and since enhanced root growth and/or delayed leaf senescence have also been implicated in plant tolerance of aridity stress, reprogramming photosynthetic cells for extended life and/or enhanced fructan biosynthesis may facilitate yield stability. and/or the plant's tolerance to abiotic stress.

[020] Accordingly, in one aspect, the present invention provides a method of manipulating fructan biosynthesis in photosynthetic cells of a plant, the method including introducing into the plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids, which encode one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.

[021] By manipulation of fructan biosynthesis, in general, we mean the increase in fructan biosynthesis in a transformed plant, in relation to a non-transformed control plant. However, for some applications, it may be desirable to reduce, or otherwise modify, fructan biosynthesis in the transformed plant, relative to the untransformed control plant. For example, it may be desirable to increase or decrease the activity of certain enzymes in the fructan biosynthetic pathway in the transformed plant relative to the untransformed control plant.

[022] By photosynthetic cells, we mean those cells in which photosynthesis occurs. In general, such cells contain the pigment chlorophyll and are otherwise known as green cells. Most photosynthetic cells are contained in plant leaves. Preferably, the genetic construct of the present invention is expressed in bundle sheath cells, more preferably in mesophilic and/or parenchymal bundle sheath cells.

[023] By an effective amount, we mean an amount sufficient to result in an identifiable phenotypic characteristic in the plant, or in a plant, plant seed or other plant part derived therefrom. Such quantities can be readily determined by a technician approximately specialized in the subject, taking into account the type of plant, the route of administration and other relevant factors. Such a technician will be readily able to determine a suitable amount and method of administration. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

[024] By genetic construct, we mean a recombinant nucleic acid molecule.

[025] A promoter means a nucleic acid sequence sufficient for direct transcription of an operably linked nucleic acid sequence.

[026] By operatively linked, it is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to allow the expression of the nucleic acid(s) under appropriate conditions, for example, when appropriate molecules such as transcriptional activator proteins are linked to the regulatory sequence. Preferably, an operably linked promoter is upstream of the associated nucleic acid.

[027] By upstream, we mean in the 3' to 5' direction along the nucleic acid.

[028] Nucleic acid means a chain of nucleotides capable of carrying genetic information. The term, in general, refers to genes, or functionally active fragments or variants thereof and/or other sequences in the organism's genome, which influence its phenotype. The term nucleic acid includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA), which are single- or double-stranded, optionally containing synthetic, unnatural, or altered nucleotide bases, synthetic nucleic acids, and combinations thereof. same.

[029] By a nucleic acid encoding a fructan biosynthetic enzyme, we mean a nucleic acid encoding a fructan biosynthetic pathway enzyme in plants, for example, fructosyl transferases, such as sucrose: sucrose 1-fructosyl transferase (1-SST); fructan: fructan 1-fructosyltransferase (1-FFT); sucrose: fructan 6-fructosyl-transferase (6-SFT) and fructan: fructan 6G-fructosyl-transferase (6G-FFT); and fructohexohydrolases, such as 1-fructohexohydrolase (1-FEH) and 6-fructohexohydrolase (6-FEH).

[030] By functionally active fragment or variant, in relation to a promoter, it is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of directing the transcription of an operably linked nucleic acid. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated, as long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least approximately 80% activity with respect to the relevant part of the sequence mentioned above to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Preferably, the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 300 nucleotides.

[031] By functionally active(s), in relation to the nucleic acid encoding a fructan biosynthetic enzyme, it is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating fructan biosynthesis in a plant by the method of the present invention, for example, being translated into an enzyme, which is capable of participating in the fructan biosynthetic pathway. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated, as long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least approximately 80% activity with respect to the relevant part of the sequence mentioned above to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.

[032] By changes in conservative nucleic acids, we mean replacements of nucleic acids that result in the conservation of the amino acid in the encoded protein, due to the degeneration of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes that result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

[033] By conservative amino acid substitutions, we mean the replacement of one amino acid with another of the same class, the classes being as follows: Non-polar: Ala, Vai, Leu, lie, Pro, Met, Phe, Trp Charged polar : Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acids: Asp, GIu Basics: Lys, Arg, His

[034] Other conservative amino acid substitutions can also be made as follows: Aromatics: Phe, Tyr, His Proton donors: Asn, Gin, Lys, Arg, His, Trp Proton acceptors: GIu, Asp, Thr, Ser, Tyr, Asn, Gin

[035] Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17,18 and 38 herein, for example, (N/S)DP(N)G, FR.DP and EC(I)D.

[036] Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in LoHum FT, invertase and FEH sequences, for example, as shown in Figures 17, 18 and 36 here.

[037] Preferably, the fragment has a size of at least 20 nucleotides, more preferably, at least 50 nucleotides, more preferably, at least 100 nucleotides, more preferably, at least 200 nucleotides, more preferably, at least 500 nucleotides.

[038] Preferably, the nucleic acid encoding one or more fructan biosynthetic enzymes is selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G-FFT, combinations thereof, and functionally active fragments and variants thereof. Preferably, the nucleic acid encodes a TF fusion protein of two or more such fructan biosynthetic enzymes.

[039] Even more preferably, the nucleic acid, which encodes one or more fructan biosynthetic enzymes, encodes 1-SST and/or 6G-FFT, even more preferably a TF fusion protein of 1-SST and 6G-FFT, or functionally active fragments and variants thereof.

[040] Preferably, the nucleic acid, which encodes one or more fructan biosynthetic enzymes, is isolated from or corresponds to a gene or genes from a species of interest. More preferably, the gene or genes are from a species of ryegrass, fescue or wheat, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue, red fescue, bread corn and durum wheat. Even more preferably, the nucleic acid, which encodes one or more fructan biosynthetic enzymes, is isolated from or corresponds to a gene from a LoHum species, such as LoHum perenne or LoHum arundinaceum.

[041] Suitable nucleic acids, which encode fructan biosynthetic enzymes, are described in PCT/AUO1/00705 and PCT/AUO1/01275, the complete disclosures of which are incorporated herein by reference.

[042] In a particularly preferred embodiment, the nucleic acid, which encodes 1-SST, includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AUO1/00705; and the nucleotide sequences, which encode the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AUO1/00705; and functionally active fragments and variants thereof.

[043] In a particularly preferred embodiment, the nucleic acid, which encodes 6G-FFT, includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AUO1/01275, and in Figure 7 here ; and the nucleic acid sequences, which encode the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AUOl/01275; and in Figure 8 here; and functionally active fragments and variants thereof.

[044] In a particularly preferred embodiment, the nucleic acid, which encodes 1-FFT, includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AU01/00705, SEQ ID NOS: 103 and 105-109 of PCT/AUOl/01275 and in Figure 9 here; and the nucleotide sequences, which encode the polypeptide sequences shown in SEQ ID NO: 4 of PCT/AUO 1/00705, SEQ ID NO: 104 of PCT/AUO1/01275 and in Figure 10 herein; and functionally active fragments and variants thereof.

[045] Applicants have found that by generating a translational fusion of two FT genes as a single open reading frame, e.g., sucrose-sucrose 1-fructosyl-transferase (Lpl-SST) and fructan-fructan 6G-fructosyl -transferase (Lp6G-FFT) from LoHum perenne, a single mRNA transcript is produced, which is translated as a single protein, with combined enzyme activities. By expressing a translational fusion of two FT genes (e.g., Lpl-SST and Lp6G-FFT), problems associated with differences in the expression patterns of these two genes, independently integrated into the plant genome, can be alleviated, resulting in conversion of sucrose into low and high GP fructans.

[046] In a particularly preferred embodiment, the nucleic acid, which encodes the 1-SST FT and 6G-FFT fusion protein, includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 here, and the nucleic acid sequences, which encode the polypeptide sequences shown in Figures 13 and 15 herein; and functionally active fragments and variants thereof.

[047] In a particularly preferred embodiment, the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31, 32, 35, 38 and 41 to 47 here; and functionally active fragments and variants thereof.

[048] In a further aspect, the present invention provides a method of enhancing the productivity of a biochemical pathway in a plant, the method including introducing, into the plant, an effective amount of a genetic construct including nucleic acids, which encode two or more enzymes from the pathway, or functionally active fragments or variants thereof.

[049] Preferably, the nucleic acids are linked to form a fusion gene, which encodes a fusion protein of the two or more enzymes.

[050] By a biochemical pathway, we mean a plurality of chemical reactions that are catalyzed by more than one enzyme or enzyme subunit and that result in the conversion of a substrate into a product. This includes, for example, a situation in which two or more enzyme subunits (each a discrete protein encoded by a separate gene) combine to form a processing unit that converts a substrate into a product. A biochemical pathway is not constrained by temporal or spatial sequentiality.

[051] By productivity intensification, in general, it is understood that the amount of product from the biochemical pathway, or product production rate, is increased in a transformed plant, in relation to a non-transformed control plant. However, for some applications, it may be desirable to reduce, or otherwise modify, the amount of biochemical pathway product or the rate of product production in the transformed plant, relative to the untransformed control plant. For example, it may be desirable to increase or decrease the amount of a pathway intermediate, or its production rate, in a transformed plant, relative to an untransformed control plant.

[052] By a fusion protein, we mean a hybrid or chimeric protein produced in a recombinant manner, by expression of a fusion gene including two or more linked nucleic acids, which originally encode separate proteins, or fragments or variants functionally active.

[053] By a fusion, translational fusion or fusion gene, we mean that two or more nucleic acids are linked in such a way as to allow expression of the fusion protein, preferably as a translational fusion. Typically, this involves removing the stop codon from a nucleic acid sequence encoding a first protein, then attaching the nucleic acid sequence from a second protein into frame. The FT fusion gene is then expressed by a cell as a single protein. The protein can be engineered to include the complete sequence of both original proteins, or a functionally active fragment or variant of each or both.

[054] The genetic construct may also include a nucleic acid sequence, which encodes a linker between the two linked nucleic acids. A linker is any chemical, synthetic, carbohydrate, lipid, polypeptide molecule (or combinations thereof) positioned between and joined to two adjacent active fragments in a fusion protein. A preferred linker of the invention is a flexible linker, such as a polypeptide chain consisting of one or more amino acid residues linked by amino acid bonds to the two active fragments. For example, a (Gly4 Sera) linker can be positioned between the two active fragments in the fusion protein.

[055] By functionally active(s), in relation to nucleic acids, which encode two or more enzymes from a biochemical pathway, it is understood that the fragment or variant (such as an analogue, derivative or mutant) is capable of intensify the productivity of the biochemical pathway in a plant, by the method of the present invention. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated, as long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least approximately 80% activity with respect to the relevant part of the sequence mentioned above to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes. By changes in conservative nucleic acids, we mean replacements of nucleic acids that result in the conservation of the same amino acid in the encoded protein, due to the degeneration of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes that result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. By conservative amino acid substitutions, we mean the replacement of one amino acid with another of the same class, the classes being as follows: Non-polar: Ala, Val, Leu, Phe, Pro, Met Phe, Trp Non-charged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acids: Asp, GIu Basics: Lys, Arg, His

[056] Other conservative amino acid substitutions can also be made as follows: Aromatics: Phe, Tyr, His Proton donors: Asn, Gin, Lys, Arg, His, Trp Proton acceptors: GIu, Asp, Thr, Ser, Tyr, Asn, Gin

[057] Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17,18 and 38 herein, for example, (N/S)DP(N)G, FR.DP and EC(I)D.

[058] Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in FT, invertase and LoHum FEH sequences, for example, as shown in Figures 17, 18 and 36 here.

[059] Preferably, the fragment has a size of at least 20 nucleotides, more preferably, at least 50 nucleotides, more preferably, at least 100 nucleotides, more preferably, at least 200 nucleotides, more preferably, at least 500 nucleotides.

[060] Preferably, the biochemical pathway is a fructan biosynthetic pathway.

[061] Preferably, the two or more enzymes from the pathway are selected from the group consisting of enzymes from the fructan biosynthetic pathway in plants, for example, fructosyl transferases such as sucrose: sucrose 1-fructosyl transferase ( 1- SST); fructan: fructan 1-fructosyltransferase (1-FFT); sucrose: fructan 6-fructosyltransferase (6-SFT) and fructan: fructan 6G-fructosyltransferase (6G-FFT); and fructohexohydrolases, such as 1-fructohexohydrolase (1-FEH) and 6-fructohexohydrolase (6-FEH).

[062] Even more preferably, the nucleic acids encoding a TF fusion protein include two or more nucleic acids selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G-FFT , and functionally active fragments and variants thereof, linked to form a FT fusion gene. The nucleic acids optionally are connected by a linker, such as a flexible linker.

[063] Even more preferably, the nucleic acids, which encode a TF fusion protein, include two or more nucleic acids linked to form a TF fusion protein of 1-SST and 6G-FFT, or functionally active fragments or variants thereof, optionally connected by a connector, such as a flexible connector.

[064] Preferably, the genes, which encode enzymes of the fructan biosynthetic pathway, are isolated from or correspond to genes from a species of ryegrass or fescue, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the genes, which encode enzymes of the fructan biosynthetic pathway, are isolated from or correspond to genes from a LoHum species, such as LoHum perenne or LoHum arundinaceum.

[065] Suitable nucleic acids, which encode fructan biosynthetic enzymes, are described in PCT/AUO 1/00705 and PCT/AUOl/01275, the complete disclosures of which are incorporated herein by reference.

[066] In a particularly preferred embodiment, the nucleic acid, which encodes 1-SST, includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AUOl/00705; and the nucleotide sequences, which encode the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AUOl/00705; and functionally active fragments and variants thereof.

[067] In a particularly preferred embodiment, the nucleic acid, which encodes 6G-FFT, includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AUOl/01275, and in Figure 7 here ; and the nucleic acid sequences, which encode the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AUOl/01275; and in Figure 8 here; and functionally active fragments and variants thereof.

[068] In a particularly preferred embodiment, the nucleic acid, which encodes 1-FFT, includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AUOl/00705, SEQ ID NOS: 103 and 105-109 of PCT/AUOl/01275 and in Figure 9 here; and the nucleotide sequences, which encode the polypeptide sequences shown in SEQ ID NO: 4 of PCT/AUOl/00705, SEQ ID NO: 104 of PCT/AUOl/01275 and in Figure 10 herein; and functionally active fragments and variants thereof.

[069] In a particularly preferred embodiment, the nucleic acid, which encodes 1-SST and 6G-FFT, includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 here, and the acid sequences nucleic, which encode the polypeptide sequences shown in Figures 13 and 15 herein; and functionally active fragments and variants thereof.

[070] In a particularly preferred embodiment, the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31, 32, 35, 36, 38 and 41 to 475 here; and functionally active fragments and variants thereof.

[071] The promoter used in the constructs and methods of the present invention can be a constitutive, tissue-specific or inducible promoter. In a preferred embodiment, the promoter is a light-regulated promoter, more preferably a photosynthetic promoter. By a light-regulated promoter is meant a promoter capable of mediating gene expression in response to light stimulation. By a photosynthetic promoter is meant a promoter capable of mediating the expression of a gene encoding a protein involved in a photosynthetic pathway in plants.

[072] Less fructan accumulates in mature leaf blades than in leaf sheaths and stems. In order to specifically increase the level of fructans in leaf blades, a strategic approach has been envisioned, which expresses, in a coordinated manner, fructan biosynthesis genes in photosynthetic cells (Figure 1). The use of light-regulated or photosynthetic promoters can provide the following advantages: • Photosynthetic promoters are active in a large group of cells, including leaf blades, upper stem and outer (>55% of cells); • They are active in cells that produce sucrose (mesophilic and parenchymal bundle sheath cells); • Its expression pattern overlaps, temporally and spatially, with the accumulation of sucrose; • Fructosyl transferase activity will remove sucrose from the source, thereby preventing feedback suppression in photosynthesis, and may facilitate increases in CO2 fixation.

[073] Particularly preferred light-regulated promoters include a ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) small subunit promoter and a chlorophyll a/b binding protein (CAB) promoter, and fragments and variants functionally their assets.

[074] The light-regulated promoter can be from any suitable plant species, including monocot plants [such as corn, rice, wheat, barley, sorghum, sugar cane, pasture grasses, bioenergy grasses], dicots [such as Arabidopsis, soybeans, canola, cotton, alfalfa and tobacco] and gymnosperms.

[075] Preferably, the light-regulated promoter is isolated from or corresponds to a promoter from a species of ryegrass or fescue, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the light-regulated promoter is isolated from or corresponds to a promoter from a LoHum species, such as LoHum perenne or LoHum arundinaceum.

[076] In another embodiment, preferably, the light-regulated promoter is isolated from or corresponds to a promoter from Arabidopsis, even more preferably, Arabidopsis thaiiana.

[077] In a particularly preferred embodiment, the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 5 here, and functionally active fragments and variants thereof.

[078] In a particularly preferred embodiment, the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 38 here, and functionally active fragments and variants thereof.

[079] In another particularly preferred embodiment, the CAS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 4 here, and functionally active fragments and variants thereof.

[080] In another particularly preferred embodiment, the promoter may be a constitutive promoter, such as a ubiquitin promoter (Ubi).

[081] In a particularly preferred embodiment, the Ubi promoter includes a sequence selected from the group consisting of the sequence shown in Figure 41 here, and functionally active fragments and variants thereof.

[082] The genetic constructs of the present invention can be introduced into plants by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells (for example, by transduction, transfection, transformation or genetic targeting) are well known to those skilled in the art. Such techniques include Agrobacterium-mediated introduction, Rhizobium-mediated introduction, electroporation into tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injections into premature embryos, and introduction by high-velocity projectiles into cells, tissues, calluses, immature embryos. and mature, ballistic transformation, Whisker transformation, and combinations thereof. The choice of technique will largely depend on the type of plant to be transformed, and can be readily determined by an appropriately specialized technician.

[083] Cells incorporating the genetic constructs of the present invention can be selected, as described below, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be evident to the technician specialized in the subject. The resulting plants can be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

[084] The methods of the present invention can be applied to a variety of plants, including monocotyledons [such as grasses (e.g., pasture, peat and bioenergy grasses, including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, canary seed , large broom corn, cordgrass, napiergrass, wildrye, wild sugar cane, Miscanthus, switchgrass), corn, rice, wheat, barley, sorghum, sugar cane, rye, oats)], dicotyledons [ such as Arabidopsis, tobacco, soybeans, canola, alfalfa, potatoes, cassava, clovers (e.g., white clover, red clover, subterranean clover), Brassica vegetables, lettuce, spinach] and gymnosperms.

[085] In another aspect of the present invention, there is provided a genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of a plant, the genetic construct, including a light-regulated promoter, or functionally active fragment or variant thereof, operatively linked to nucleic acids, which encode one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.

[086] In yet another aspect of the present invention, there is provided a genetic construct capable of enhancing the productivity of a biochemical pathway in a plant, the genetic construct including nucleic acids, which encode two or more enzymes of the pathway, or fragments or variants functionally active of them.

[087] Preferably, the nucleic acids are linked to form a fusion gene, which encodes a fusion protein of the two or more enzymes.

[088] In preferred embodiments, genetic constructs, in accordance with various aspects of the present invention, may be vectors.

[089] By a vector, we mean a genetic construct used to transfer genetic material to a target cell.

[090] The vector may be of any suitable type and may be viral or non-viral. The vector can be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g., derived from plant viruses; bacterial plasmids; Ti plasmid derivatives from Agrobacteríum tumefaciens-, Ri plasmid derivatives from Agrobacteríum rhizogenes-, phage DNA; yeast artificial chromosomes; artificial chromosomes from bacteria; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector can be used, as long as it is replicable or integrative or viable in the plant cell.

[091] In a preferred embodiment of this aspect of the invention, the genetic construct may additionally include a terminator; the promoter, the gene and the terminator being operatively linked.

[092] The promoter, gene and terminator can be of any suitable type and can be endogenous in relation to the target plant cell or can be exogenous, as long as they are functional in the target plant cell.

[093] A variety of terminators, which can be used in the genetic constructs of the present invention, are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or from a different gene. Particularly suitable terminators are polyadenylation signals such as (CaMV)35S polyA and other terminators from the nopaline synthase (nos) and octopine synthase (ocs) genes.

[094] The genetic construct, in addition to the promoter, the gene and the terminator, may include additional elements necessary for the expression of the nucleic acid, in different combinations, for example, vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize ubiquitin Ubi intron), antibiotic resistance genes, and other selectable marker genes [such as the neomycin phosphotransferase gene (nptll), the hygromycin gene phosphotransferase (hph), the phosphinothricin acetyltransferase gene (bar or pat), and reporter genes (such as the betaglucoronidase (GUS) gene (gusA)]. The genetic construct may also contain a ribosome binding site for the initiation of translation. The genetic construct may also include appropriate sequences to amplify expression.

[095] In particular, the genetic construct may additionally include a nucleic acid sequence, which encodes a linker between the two linked nucleic acids, as previously described.

[096] Those skilled in the art will appreciate how the various components of the genetic construct are operatively linked to result in expression of the nucleic acid. Techniques for operatively linking the components of the genetic construct of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example, including one or more restriction enzyme sites.

[097] Preferably, the genetic construct is substantially isolated or purified. By substantially purified, it is meant that the genetic construct is free of genes, which, in the naturally occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter. Therefore, the term includes, for example, a genetic construct, which is incorporated into a vector; on a plasmid or autonomously replicating virus; or in the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a genetic construct, which is part of a hybrid gene, which encodes additional polypeptide sequence. Preferably, the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.

[098] The term isolated means that the material is removed from its natural environment (for example, the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated by the fact that such a vector or composition is not part of its natural environment.

[099] As an alternative to using a selectable marker gene, to provide a phenotypic characteristic for selection of transformed host cells, the presence of the genetic construct in transformed cells can be determined by other techniques well known in the art, such as PCR (acronym, in English, for polymerase chain reaction), Southern blot hybridization analysis, histochemical assays (e.g., GUS assays), thin layer chromatography (TLC), Northern and Western blot hybridization analyzes

[0100] The applicant has also found that the methods of the present invention can result in increased biomass in the transformed plant, relative to a non-transformed control plant. This enhanced biomass can, in turn, be used as a screening tool for identifying transformed plants. This has the advantage that in some circumstances it may be necessary to include an antibiotic resistance or other marker to select transformants, where subsequent removal of such markers (and the creation of marker-free plants) may present difficulties.

[0101] By biomass intensification or intensified biomass, we mean intensification of, increase in, or increased stability of biomass yield, including shoot and/or root growth, in a transformed plant, relative to a non-transformed control plant. transformed. For example, one or more growth traits selected from the group consisting of plant height, pasture dry weight, total leaf area, cumulative leaf area, leaf growth dynamics (i.e., the number of leaves along of time), number of shoots, number of shoots, number of roots, mass or weight of roots, mass or weight of shoots, length of roots, length of shoots, length of runners, number of tubers, length of tubers, number of flowers, number of fruits, number of seeds, seed weight, fruit weight, percentage of flowering plants and seed yield per flower or per sown area; may be enhanced, increased, or more stable in a transformed plant relative to an untransformed control plant.

[0102] This technique is particularly applicable to plants that are substantially genetically uniform or genetically identical or that exhibit small differences in phenotype in biomass, prior to transformation.

[0103] Accordingly, in another aspect of the present invention, there is provided a method of enhancing biomass in a plant, the method including introducing into the plant an effective amount of a genetic construct including a promoter, or a fragment or functionally active variant thereof, operatively linked to nucleic acids, which encode one or more fructan biosynthetic enzymes, or a functionally active fragment or variant thereof. Preferably, the promoter is a light-regulated promoter.

[0104] In yet another aspect of the present invention there is provided a method of increasing biomass in a plant, the method including introducing into the plant an effective amount of a genetic construct including nucleic acids, which encode two or more enzymes. from a biochemical pathway in the plant, or functionally active fragments or variants thereof.

[0105] In yet another aspect of the present invention there is provided a method of increasing biomass in a plant, the method including introducing into the plant effective amounts of a genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of the plant. plant, and a genetic construct capable of manipulating senescence in the plant.

[0106] The genetic constructs can be introduced into the plant by any suitable technique, as previously described, and can be introduced concurrently, sequentially or separately.

[0107] Preferably, the genetic construct capable of manipulating fructan biosynthesis includes a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids, which encode one or more fructan biosynthetic enzymes, or fragments or functionally active variants thereof. Preferably, the promoter is a light-regulated promoter.

[0108] Preferably, the genetic construct capable of manipulating senescence in the plant is capable of manipulating senescence in photosynthetic cells of the plant.

[0109] Preferably, the genetic construct, capable of manipulating senescence, includes a MYB gene promoter or modified MYB gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene, which encodes a enzyme involved in the biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.

[0110] Suitable genetic constructs or vectors are described in international patent application number PCT/AUOl/01092 and in US patent application number 11/789,526, the complete disclosures of which are incorporated herein by reference.

[0111] Senescence manipulation, in general, refers to the delay of senescence in the transformed plant or cells or organs of the transformed plant, e.g., photosynthetic cells, relative to a non-transformed control plant. However, for some applications it may be desirable to promote, or otherwise modify, senescence in the plant. Senescence can be promoted, or otherwise modified, for example, by using a nonsense gene.

[0112] The MYB gene promoter can be of any suitable type. Preferably, the MYB gene promoter is a MYB32 gene promoter. Preferably, the MYB gene promoter is from Arabidopsis, more preferably Arabidopsis thaiiana. Most preferably, the MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1 of PCT/AUO1/01092 and functionally active fragments and variants thereof.

[0113] A suitable promoter is described in Li et al., Cloning of three MYB-iike genes from Arabidopsis (PGR 99-138) Plant Physiology 121:313 (1999), the full disclosure of which is incorporated herein by reference.

[0114] By a modified MYB gene promoter is meant a promoter normally associated with a MYB gene, which promoter is modified to eliminate or inactivate one or more root-specific motifs and/or pollen-specific motifs in the promoter.

[0115] Preferably, the modified MYB gene promoter is a modified MYB32 gene promoter. Preferably, the modified MYB gene promoter is modified from the MYB gene promoter from Arabidopsis, or more preferably, Arabidopsis thaiiana.

[0116] A suitable promoter, which can be modified in accordance with the present invention, is described in Li et al., Cloning of three MYB-iike genes from Arabidopsis (PGR 99-138) Plant Physiology 121:313 (1999), the full disclosure of which is incorporated herein by reference.

[0117] By a root-specific motif is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 5 nucleotides, that directs the expression of any associated gene in the roots of a plant.

[0118] Preferably, the root-specific motif includes an ATATT or AATAT consensus sequence.

[0119] By a pollen-specific motif is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 4 or 5 nucleotides, that directs the expression of an associated gene in the pollen of a plant .

[0120] Preferably, the pollen-specific motif includes a consensus sequence selected from the group consisting of TTTCT, AGAAA, TTCT and AGAA.

[0121] A specific root or pollen motif can be inactivated by addition, deletion, substitution or derivatization of one or more nucleotides within the motif so that it no longer has the preferred consensus sequence.

[0122] Preferably, the modified MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOS: 2, 3 and 4 of US document number 11/789,526 and fragments and variants functionally active of them.

[0123] By a gene, which encodes an enzyme involved in the biosynthesis of a cytokinin, is meant a gene, which encodes an enzyme involved in the synthesis of cytokinins, such as kinetin, zeatin and benzyladenine, for example, a gene, encoding isopentyl transferase (IPT), or a gene similar to IPT, such as the sho gene (e.g., petunia). Preferably, the gene is an isopentyl transferase (IPT) gene or sho gene. In a preferred embodiment, the gene is from a species selected from the group consisting of Agrobacterium, more preferably Agrobactehum tumefaciens, Lotus, more preferably Lotus japonicus, and Petunia, more preferably Petunia hybrida.

[0124] Most preferably, the gene includes a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOS: 5, 7 and 9 of US document number 11/789,526, sequences, which encode the polypeptides shown. in SEQ ID NOS: 6, 8 and 10 of North American document number 11/789,526, and functionally active fragments and variants thereof.

[0125] The present invention also provides a method of selection for transformed plants, the method including introducing, into the plants, an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, linked in a manner operative to nucleic acids, which encode one or more fructan biosynthetic enzymes, or functionally active fragment or variants thereof, and the selection of plants with enhanced biomass. Preferably, the promoter is a light-regulated promoter.

[0126] In a further aspect of the present invention, there is provided a transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics or enhanced biomass, relative to an untransformed control plant.

[0127] By modified fructan biosynthetic characteristics, it is meant that the transformed plant exhibits increased fructan biosynthesis and/or contains increased levels of soluble carbohydrate, relative to an untransformed control plant.

[0128] In a preferred embodiment, the transgenic plant cell, plant, plant seed or other biomass-enhanced plant part has an increase in biomass of at least approximately 10%, more preferably at least approximately 20%, more preferably , at least approximately 30%, more preferably, at least approximately 40%, relative to an untransformed control plant.

[0129] For example, biomass can be increased by between approximately 10% and 300%, more preferably, between approximately 20% and 200%, more preferably, between approximately 30% and 100%, more preferably, between approximately 40% and 80%, in relation to an untransformed control plant.

[0130] For example, plant height can be increased by between approximately 10% and 300%, more preferably, between approximately 20% and 200%, more preferably, between approximately 30% and 100%, more preferably, between approximately 40%. % and 80%, in relation to an untransformed control plant.

[0131] For example, the dry weight of pasture can be increased by between approximately 10% and 600%, more preferably, between approximately 20% and 400%, more preferably, between approximately 30% and 300%, more preferably, between approximately 40% and 200%, in relation to an untransformed control plant.

[0132] In a preferred embodiment, the transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics has an increase in soluble carbohydrate of at least approximately 10%, more preferably, of at least approximately 20%. %, more preferably at least approximately 30%, more preferably at least approximately 40%, relative to an untransformed control plant.

[0133] For example, soluble carbohydrates can be increased by between approximately 10% and 300%, more preferably, between approximately 20% and 200%, more preferably, between approximately 30% and 100%, more preferably, between approximately 40% and 80%, in relation to an untransformed control plant.

[0134] For example, the fructan concentration can be increased between approximately 10% and 600%, more preferably, between approximately 20% and 400%, more preferably, between approximately 30% and 200%, more preferably, between approximately 40% and 150%, in relation to an untransformed control plant.

[0135] Preferably, the plant cell, plant, plant seed or other plant part includes a genetic construct or vector, according to the present invention. Preferably, the transgenic plant cell, plant, plant seed or other plant part is produced by a method in accordance with the present invention.

[0136] The present invention also provides a transgenic plant, plant, plant seed or other plant part derived from a plant cell of the present invention and including a genetic construct or vector of the present invention.

[0137] The present invention also provides a transgenic plant, plant seed or other plant part derived from a plant of the present invention and including a genetic construct or vector of the present invention.

[0138] Preferably, the transgenic plant cell, plant, plant seed or other plant part is a species of LoHum, more preferably LoHum perenne or LoHum arundinaceum. Preferably, the transgenic plant cell, plant, plant seed or other plant part is a cereal grain, more preferably a species of Triticum, most preferably wheat (Triticumaestivum).

[0139] For example, the present invention enables the production of transgenic perennial ryegrass plants, with increased fructans in leaf blades, vigorous growth and/or greater tolerance to abiotic stress, for improved nutrition for grazing animals.

[0140] The present invention also makes it possible to produce transgenic wheat plants with increased fructans, vigorous growth and/or tolerance to abiotic stress, for increased mass of useful carbohydrates, for example, for the production of biofuels or animal feed.

[0141] A plant cell is any self-propagating cell limited by a semipermeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein, includes, without limitation, algae, cyanobacteria, seed suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

[0142] By transgenic is meant any cell that includes a DNA sequence, which is inserted by artifice into a cell and becomes part of the genome of the organism that develops from that cell. As used herein, transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into either the nuclear genome or the plastid genome.

[0143] In a further aspect of the present invention, there is provided a fusion protein comprising two or more enzymes of a biochemical pathway in a plant, or functionally active fragments or variants thereof.

[0144] By functionally active, in this context, it is meant that the fragment or variant has one or more biological properties of the corresponding protein, from which the fragment or variant is derived. Additions, deletions, substitutions and derivatizations of one or more amino acids are contemplated, as long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably, the fragment or variant has approximately 80% identity with respect to the relevant part of the sequence mentioned above to which the fragment or variant corresponds, more preferably at least approximately 90% identity, more preferably at least approximately 95% identity. % identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

[0145] Preferably, the fragment has a size of at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 50 amino acids, more preferably at least 100 amino acids, more preferably at least 200 amino acids.

[0146] Preferably, the biochemical pathway is in the fructan biosynthetic pathway.

[0147] Preferably, the two or more enzymes from the pathway are selected from the group consisting of enzymes from the fructan biosynthetic pathway in plants, for example, fructosyl transferases such as sucrose: sucrose 1-fructosyl transferase ( 1- SST); fructan: fructan 1-fructosyltransferase (1-FFT); sucrose: fructan 6-fructosyltransferase (6-SFT) and fructan: fructan 6G-fructosyltransferase (6G-FFT); and fructohexohydrolases, such as 1-fructohexohydrolase (1-FEH) and 6-fructohexohydrolase (6-FEH).

[0148] Even more preferably, the fusion protein is a 1-SST FT and 6G-FFT fusion protein, or functionally active fragment or variants thereof.

[0149] Preferably, the two or more enzymes from the pathway correspond to enzymes from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the two or more enzymes from the pathway correspond to enzymes from a LoHum species, such as LoHum perenne or LoHum arundinaceum.

[0150] Suitable fructan biosynthetic enzymes are described in PCT/AU01/00705 and PCT/AU01/01275, the complete disclosures of which are incorporated herein by reference.

[0151] In a particularly preferred embodiment, 1-SST includes an amino acid sequence shown in SEQ ID NO: 12 of PCT/AUO1/00705, or a functionally active fragment or variant thereof.

[0152] In a particularly preferred embodiment, the 6G-FFT includes an amino acid sequence shown in SEQ ID NO: 111 of PCT/AUO1/01275 or in Figure 8 here, or a functionally active fragment or variant thereof.

[0153] In a particularly preferred embodiment, the 1-SST_6G-FFT TF fusion protein includes an amino acid sequence shown in Figures 13 or 15 herein, or functionally active fragments or variants thereof.

DETAILED DESCRIPTION OF ACHIEVEMENTS

[0154] The present invention will now be more fully described with reference to the accompanying examples and drawings. However, it should be understood that the following description is illustrative only and should not be taken, in any way, as a restriction on the generality of the invention described above.

[0155] In the Figures:

[0156] Figure 1. Model for directed expression of fructan biosynthesis genes in photosynthetic cells in leaf blades. The expression of fructosyl transferase (FT) genes is compelled by photosynthetic promoters. Fructan biosynthesis then occurs in photosynthetic cells, which produce sucrose. Pyramiding with modification of cytokinin biosynthesis to delay leaf senescence, thereby extending the life of photosynthetic cells, which are engineered to synthesize fructans, and leading to increased biomass production.

[0157] Figure 2. Expression of the RuBisCO small subunit gene in perennial ryegrass is regulated by light, as shown by quantitative real-time RT-PCR. Tissue sampling occurred every four hours. The boxes represent periods of daylight.

[0158] Figure 3. In silico expression patterns of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (LpRbcS) and chlorophyll a/b binding protein (LpCAB), in perennial ryegrass, shows that it is the most abundant in vegetative tissues. LpRbcS (contig LPCL9_C359) is represented by the 47 ESTs and LpRbcS (contig LPCL1112_C12) is represented by the 19 ESTs.

[0159] Figure 4. Nucleotide sequences of the LpCAB promoter (SEQ ID NO: 1).

[0160] Figure 5. Nucleotide sequences of the LpRbcS promoter (SEQ ID NO: 2).

[0161] Figure 6. Schematic representation of the fructan biosynthetic pathway in some grasses.

[0162] Figure 7. Nucleotide sequence of the open reading frame of Lp6G-FFT (SEQ ID NO: 3).

[0163] Figure 8. Deduced amino acid sequence of Lp6G-FFT (SEQ ID NO: 4).

[0164] Figure 9. Nucleotide sequence of the Lpl-FFT open reading frame (SEQ ID NO: 5).

[0165] Figure 10. Deduced amino acid sequence of Lpl-FFT (SEQ ID NO: 6).

[0166] Figure 11. Schematic representation of the strategy used to generate the translational FT fusion of the fructosyl transferase genes Lpl-SST and Lp6G-FFT (Lpl-SST_Lp6G-FFT).

[0167] Figure 12. Nucleotide sequence of the open reading frame of Lpl-SST_Lp6G-FFT FT fusion 1 (SEQ ID NO: 7).

[0168] Figure 13. Deduced amino acid sequence of Lpl-SSTJ_p6G-FFT FT fusion 1 (SEQ ID NO: 8).

[0169] Figure 14. Nucleotide sequence of the open reading frame of Lpl-SST_Lp6G-FFT FT fusion 3 (SEQ ID NO: 9).

[0170] Figure 15. Deduced amino acid sequence of Lpl-SST_Lp6G-FFT FT fusion 3 (SEQ ID NO: 10).

[0171] Figure 16. Schematic representation of the strategy used to generate the different translational FT fusions of the fructosyl transferase genes Lpl-SST, Lp6G-FFT and Lpl-FFT.

[0172] Figure 17. (A) and (B) Hypothetical model of the interaction of TF proteins to form a transmembrane protein. (C) Representation of key protein domains in Lpl-SST-6G-FFT proteins. Box 1: (N/S)DPNG; Box 2: RDP and Box 3: EC represent the highly conserved domains involved in binding to and hydrolysis of substrate (sucrose). Crosses (X) represent the highly conserved amino acid sequences (domains) found among FT, invertase, and FEH sequences from LoHum species. Large subunit LS, small subunit SU. Representation of the active domains within the amino acid sequence of the Lpl-SST_Lp6G-FFT FT 3 fusion protein can be found in Figure 36.

[0173] Figure 18. Amino acid alignment of FT, INV and FEH from LoHum perenne (SEQ ID NOS: 11-33). Key protein domains, found among the FT, invertase and FEH sequences, such as (N/S)DPNG, RDP and EC, which represent the highly conserved domains involved in binding to and hydrolysis of substrate (sucrose), are underlined in bold and marked. Highly conserved amino acid domains found among the FT, invertase and FEH sequences from LoHum species are underlined. The representation of the active domains within the amino acid sequence of the FT Lpl-SST_Lp6G-FFT fusion protein 3 can be found in Figure 36.

[0174] Figure 19. Functional analysis of fructan: fructan 6G-fructosyl-transferase (Lp6G-FFT). A. Plasmid map of Lp6G-FFT in the yeast expression vector. B. Protein excreted from yeast containing either pPICZoA::Lp6G-FFT vector or pPICZoA vector separated only by polyacrylamide gel electrophoresis. C. Traces of water-soluble carbohydrate (CSA) after high-pressure anion exchange chromatography (CTAAP). CSAs were isolated from onion, or 1-kestose solution incubated with either purified Lp6G-FFT protein (pPICZoA: :Lp6G-FFT) or with control seed with vector (pPICZoA).

[0175] Figure 20. Base target vector, pPZP200-ubi:bar-nos R4 R3, used in recombinational cloning with multisite gate.

[0176] Figure 21. Outline of the procedure for the in planta transient expression system. Agrobacterium cultures are prepared, which harbor the expression constructs. These are injected into tobacco leaves. Three days after filtration, protein expression was tested. Top right panel shows GUS activity, bottom right panel shows example of water-soluble carbohydrate separation by CTAAP.

[0177] Figure 22. High performance anion exchange chromatography (CTAAD) is used to separate and quantify carbohydrates using standards (1-ketose), and to quantify the amount of total fructans extracted from a control plant (35S::GUS ) and from transgenic plants transiently overexpressing Lpl-SST (35S::1-SST), Lp6G-FFT (35S::6G-FFT) and the FT fusion (35S::Lpl-SST_Lp6G-FFT).

[0178] Figure 23. Wheat RuBisCO promoter target vectors compelling expression of (A) Lpl-SST, (B) Lp6G-FFT, (C) FT fusion 1 LplSST_Lp6GFFT, (D) FT fusion 3 LplSST_Lp6GFFT, and (E) the GUS marker gene.

[0179] Figure 24. TaRbcS:: Lpl-SST: :TaRbcS expression cassette sequence (SEQ ID NO: 34). The regulatory sequences, TaRbcS promoter and terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0180] Figure 25. TaRbcS expression cassette sequence: :Lp6GFFT::TaRbcS (SEQ ID NO: 35). The regulatory sequences, TaRbcS promoter and terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0181] Figure 26. Sequence of FT TaRbcS fusion 1 expression cassette: :Lpl- SST_Lp6G-FFT::TaRbcS (SEQ ID NO: 36). The regulatory sequences, TaRbcS promoter and terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0182] Figure 27. FT TaRbcS::Lpl- SST_Lp6G-FFT::TaRbcS fusion 3 expression cassette sequence (SEQ ID NO: 37). The regulatory sequences, TaRbcS promoter and terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0183] Figure 28. pBlueScript SK vector harboring the LpFT4 3' terminator sequence, pBS-LpFT4.

[0184] Figure 29. (A) The plasmid pBS-Lpl-SST::FT4 and (B) the plasmid pBS- LpRbcS::Lpl- SST::LpFT4.

[0185] Figure 30. (A) The plasmid pBS-LpCAB::LpFT4 and (B) the plasmid pBS-LpCAB::Lp6G- FFT::LpFT4.

[0186] Figure 31. LpRbcS expression cassette sequence: :Lpl-SST::LpFT4 (SEQ ID NO: 38). The regulatory sequences, LpRbcS promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the stozt (ATG) and stop (TAG) codons are shaded.

[0187] Figure 32. LpCAB expression cassette sequence: :Lp6G-FFT::LpFT4 (SEQ ID NO: 39). The regulatory sequences, LpRbcS promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0188] Figure 33. The Blunt-Lpl-SST_Lp6G-FFT FT PCR fusion plasmid.

[0189] Figure 34. Target vectors containing the ryegrass RuBisCO (LpRbcs) promoter driving FT fusions 1 and 3. (A) FT fusion 1 pBS-LpRbcS::Lpl-SST_Lp6G- FFT::LpFT4 and (B) FT fusion 3 pBS-LpRbcS::Lpl-SST-Lp6G-FFT::LpFT4.

[0190] Figure 35. Sequence of FT LpRbcS::Lpl-SSr_Lp6G-FFT::LpFT4 FT fusion 1 expression cassette (SEQ ID NO: 40). The regulatory sequences, LpRbcS promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0191] Figure 36. Sequence of FT fusion 3 expression cassette LpRbcS::Lpl-SST_Lp6G-FFT::LpFT4 (SEQ ID NO: 41). The regulatory sequences, LpRbcS promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the stozt (ATG) and stop (TAG) codons are shaded. The amino acid sequence is indicated in bold (SEQ ID NO: 42). The domains are highlighted as follows: boxes indicate highly conserved motifs in the family of 32 glycoside hydrolases, including invertases, fructosyl transferases and fructan exohydrolases, which are involved in substrate binding and hydrolysis: double underlining shows transmembrane domain ; and shaded boxes represent conservative domains among 32 glycoside hydrolases.

[0192] Figure 37. Target vector containing the Arabidopsis RuBisCO (AtRbcS) promoter driving FT fusion 3, FT fusion 3 pPZP200_AtRbcS::Lpl-SST_6G- FFT::nos.

[0193] Figure 38. Sequence of the FT AtRbcS::Lpl-SST-6G-FFT::nos fusion 3 expression construct (SEQ ID NO: 43).

[0194] Figure 39. Details of the pBlueScript SK(-) base vector from Promega, with the positions of the restriction endonuclease sites for cloning indicated.

[0195] Figure 40. Vector backbone used for construction of p-Ubi::Lpl- SST::35S and p- Ubi::Lp6G-FFT::35S (Ye et al., 2001).

[0196] Figure 41. Representative sequence of a constitutive promoter (Ubi) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 44). The regulatory sequences, Ubi promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0197] Figure 42. Representative sequence of a constitutive promoter ((CAMV)35S2) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 45). The regulatory sequences, promoter (CAMV)35S2 and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0198] Figure 43. Representative sequence of a constitutive promoter (RUBQ2) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 46). The regulatory sequences, RUBQ2Í promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the stozt (ATG) and stop (TAG) codons are shaded.

[0199] Figure 44. Representative sequence of a constitutive promoter (OsActl) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 47). The regulatory sequences, OsActl promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the stozt (ATG) and stop (TAG) codons are shaded.

[0200] Figure 45. Representative sequence of a tissue (tubercle) specific promoter (Cathlnh) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 48). The regulatory sequences, Cathlnh promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0201] Figure 46. Representative sequence of a stress-regulated promoter (Atrd29a) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 49). The regulatory sequences, Atrd29a promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the stozt (ATG) and stop (TAG) codons are shaded.

[0202] Figure 47. Representative sequence of a sucrose-regulated promoter (16R) combined with an FT fusion protein and a terminator sequence (SEQ ID NO: 50). The regulatory sequences, 16R promoter and LpFT4 terminator, are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.

[0203] Figure 48. Phenotypes of transgenic perennial ryegrass plant regeneration after cotransfection with the TaRbcS promoter light-regulated gene constructs (Table 1) and the pAcHl vector, with selection in hygromycin. Plants containing each of the FT TaRbcS::Lpl-SST_Lp6G-FFT::TaRbcS fusion constructs exhibit a growth advantage under in vitro culture conditions, thus allowing for their early identification and selection (rightmost column). .

[0204] Figure 49. Phenotypes of transgenic perennial ryegrass plant regeneration after cotransfection with the LpRbcS promoter light-regulated gene constructs with selection in hygromycin. The plants contain each of the FT 1/3 fusion constructs LpRbcS::Lpl-SST::LpFT4 or the LpRbcS::Lpl-SST_Lp6G-FFT::LPFT4. Plants containing the FT fusion constructs exhibit a growth advantage under in vitro culture conditions.

[0205] Figure 50. Phenotypes of mature plants under greenhouse conditions. Representative samples of transgenic perennial ryegrass plants in the vegetative stage. FT TaRbcS::Lpl-SST_Lp6G- FFT::LpFT4 fusion transgenic perennial ryegrass plants exhibit enhanced growth performance with larger leaves, enlarged shoots, increased root growth compared to control non-transgenic perennial ryegrass plants. The plants were also trimmed three weeks earlier. Close-up micrographs of leaf blades indicate an increase in leaf diameter in FT fusion transgenics.

[0206] Figure 51. Representative samples of mature transgenic perennial ryegrass plant phenotypes (4 weeks) under field conditions. FT fusion transgenic perennial ryegrass plants exhibit enhanced growth performance with larger leaves, enlarged shoots, increased root growth compared to control Lpl-SST transgenic perennial ryegrass plants.

[0207] Figure 52. Representative examples of phenotypic biomass classifications (1 - lowest biomass to 5 - highest biomass) of transgenic perennial ryegrass plants expressing FT fusion transgenes under field conditions.

[0208] Figure 53. Transgenic perennial ryegrass leaf phenotypes. Representative samples of manual sections of leaf blades at the vegetative stage. The left shows the comparison of full leaf sections, the right enlarged areas of leaf sections. Ad-Adaxial, Ab-abaxial.

[0209] Figure 54. Biochemical analysis (CTAAD) of level and composition of fructan present in FT TaRbcS::Lpl-SST_Lp6G-FFT::TaRbcS transgenic fusion 3, TaRbcS::Lpl- SST::TaRbcS perennial ryegrass plants , TaRbcS::Lp6G-FFT::TaRbcS and control perennial ryegrass plants harboring only the selectable marker (hph gene).

[0210] Figure 55. Biochemical analysis (CTAAD) of total fructans present in whole shoots of (A) fusion 1 of FTTaRbcS::Lpl-SST_Lp6G-FFT::TaRbcS, (B) fusion 3 of FT TaRbcS::Lpl-SST_Lp6G - FFT::TaRbcS FT, (C) TaRbcS::Lpl-SST::TaRbcS and (D) transgenic TaRbcS::6G- FFT::TaRbcS perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harboring only the selectable marker (hph gene).

[0211] Figure 56. Biochemical analysis (CTAAD) of 1-ketose present in whole shoots of (A) FT TaRbcS::Lpl-SST_Lp6G-FFT::TaRbcS fusion 1, (B) FT TaRbcS::Lpl fusion 3 -SST_Lp6G- FFT::TaRbcS, (C) TaRbcS::Lpl-SST::TaRbcS and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harboring only the selectable marker (hph gene).

[0212] Figure 57. Biochemical analysis (CTAAD) of sucrose present in whole shoots of (A) FT TaRbcS::Lpl-SST_Lp6G-FFT::TaRbcS fusion 1, (B) FT TaRbcS::Lpl-SST_Lp6G fusion 3 - FFT::TaRbcS, (C) TaRbcS::Lpl-SST::TaRbcS and (D) TaRbcS::6G-FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1 '), harboring only the selectable marker (hph gene).

[0213] Figure 58. Fructan levels in whole shoots and leaf blades in wild-type (control) and FT and LpRbcS "Lpl-SST transgenic perennial ryegrass plants grown under field conditions and harvested in December 2009 .

[0214] Figure 59. Fructan composition in leaf blades of wild-type and transgenic LpRbcS::Lpl-SST perennial ryegrass plants grown under field conditions. Box 1 represents low GP fructans (GP up to 10-15). Box 2 represents high GP fruit (GP greater than 10-15).

[0215] Figure 60. Transgene expression in full shoots of LpRbcS FT fusion and LpRbcS::Lpl-SST transgenic perennial ryegrass plants grown under field conditions. Samples were collected in November (white bars) and December (black bars) 2009. Samples were normalized against endogenous histone expression and are presented as number of copies transcribed per 35 ng of RNA.

[0216] Figure 61. Phenotypic analysis of transgenic perennial ryegrass after propagation for 7 weeks (A-C) and for 12 weeks (D-E) in pot mixture from a single shoot. Fusion 1 of FTTarbcS::Lpl- SST_Lp6G-FFT::Tarbcs (A, D) and fusion 3 of FT TarbcS::Lpl-SST_Lp6G-FFT::Tarbcs (B) plants exhibit increased leaf length and number of shoots in plants fusion compared to control plants expressing only the hph gene (C, E).

[0217] Figure 62. Quantitative phenotypic analysis of FT TarbcS::Lpl-SST_Lp6G- FFT::Tarbcs transgenic and FT TarbcS::Lpl-SST_Lp6G-FFT::Tarbcs fusion 3 plants after growth for 7 weeks (white bars) and for 12 weeks (black bars). Measurements were conducted for plant height (A), leaf width (B) and number of shoots (C) compared to the average of 8 control plants expressing only the hph gene.

[0218] Figure 63. Transgenic perennial ryegrass plants expressing FT fusion 3 (AtMYB32::IPT) only from LXR® technology, only FT fusion 3 LpRbcS::Lpl- SST_Lp6G-FFT::LpFT4, as well as LXR® and fusion 3 of FT LpRbcS::Lpl-SST_Lp6G- FFT::LpFT4 FT together under greenhouse conditions.

[0219] Figure 64. Pasture dry weight analysis of GOI-ve control perennial ryegrass plants (average of five lines) and independent FT fusion only or transgenic FT plus LXR® fusion grown under greenhouse conditions and collected 6 weeks post-pruning.

[0220] Figure 65. Fructan levels in leaf blades of GOI-ve control (average of five lines) and independent FT fusion-only or transgenic FT plus LXR® fusion perennial ryegrass plants, grown under conditions of stove.

[0221] Figure 66. Transgenic tall fescue plants expressing FT LpRbcS::Lpl-SST_Lp6G- FFT::LpFT4 fusion 3 under greenhouse conditions.

[0222] Figure 67. Pasture dry weight analysis of tall fescue plants grown in a GOI-ve control greenhouse (average of five lines) and independent FT fusion only or transgenic FT plus LXR® fusion.

[0223] Figure 68. Number of shoots of tall fescue plants grown in a GOI-ve control greenhouse (average of five lines) and independent FT fusion only or transgenic FT plus LXR® fusion.

[0224] Figure 69. Accumulation of fructan in leaf blades of tall fescue lines grown in a GOI-ve control greenhouse (average of five lines) and independent transgenics that express the FT fusion.

[0225] Figure 70. Plant regeneration phenotypes of transgenic wheat plants after transformation with the light-regulated gene constructs. Transgenic wheat plants growing in vitro that contain the FT Lpl-SST_Lp6G-FFT fusion construct exhibit a growth advantage under in vitro conditions, thus enabling their early identification and selection. The superior growth phenotype of transgenic wheat TF fusion lines was observed during the early stages of in vitro plant regeneration conducted in tissue culture plates. Six weeks after incubation under light conditions, the calli exhibited additionally developed in vitro growing shoots/shoots (panel A) and more specifically additionally developed in vitro growing roots (panel B) in the in vitro growing transgenic wheat plants, which contain the Lpl-SST_Lp6G- FFT FT fusion construct, compared to control plants.

[0226] Figure 71. Transgenic wheat plants, which contain the TaRbcS::Lpl-SST_Lp6G- FFT::TaRbcS FT fusion construct, exhibited an obvious early increase in shoot number when compared to control plants growing under (A) 2 months under in vitro conditions and (B) under greenhouse conditions.

[0227] Figure 72. Transgenic wheat plants, which contain FT fusion constructs, exhibited an obvious early increase in shoot number when compared to control plants grown under greenhouse conditions.

[0228] Figure 73. Transgenic wheat plants, which contain LXR® technology, exhibited an obvious early increase in shoot number when compared to control plants under greenhouse conditions (A). They also exhibited an increase in photosynthetic tissue after 35 days under greenhouse conditions (B).

[0229] Figure 74. Phenotypic analysis of transgenic wheat plants, which express only LXR® technology (AtMYB3::IPT::35S), only FT fusion 3 TaRbcS: :Lpl- SST_Lp6G-FFT::TaRbcS, as well as LXR ® and FT fusion 3 TaRbcS: :Lpl-SST_Lp6G- FFT::TaRbcS together under oven conditions.

[0230] Figure 75. Fructan accumulation and shoot number in transgenic wheat plants containing or only FT or LXR® fusion constructs plus FT fusion constructs, when compared to controls transformed with genes of less interest (GOI-) .

[0231] Figure 76. Fructan accumulation in control wheat plants TI GOI-ve, FT fusion only and LXR® plus transgenic FT fusion. nine weeks after sowing. The fructan level in the control represents the average of data obtained from six GOI-ve plants.

[0232] Figure 77. Phenotype of transgenic white clover plants expressing FT LXR® fusion constructs, FT fusion constructs AtRbcS::Lpl- SST-6G_FF::nos or LXR® plus AtRbcS::Lpl-SST- 6G_FF::nos, when compared to the less transformed GOI controls.

[0233] Figure 78. Transgenic expression levels of the FT fusion transgene driven by the AtRbcS promoter in white clover plants. Controls were wild-type plants. Samples were normalized against endogenous histone expression and are presented as number of copies transcribed per 35 ng of RNA.

[0234] Figure 79. Fructan accumulation in wild-type control, AtRbcS FT fusion and AtRbcS plus LXR® transgenic fusion white clover lines.

[0235] Figure 80. Phenotype of Arabidopsis plants expressing LXR®, FT fusion AtRbcS::Lpl-SST-6G_FF::nos or LXR® plus FT fusion AtRbcS::Lpl-SST-6G_FF constructs ::us, when compared to the less transformed GOI controls.

[0236] Figure 81. Transgenic expression levels of the FT fusion transgene driven by the AtRbcS promoter in Arabidopsis plants. Controls were wild-type plants. Samples were normalized against endogenous histone expression and are presented as number of copies transcribed per 35 ng of RNA.

[0237] Figure 82. Transgenic FT T2 fusion Arabidopsis plants grown in soil.

[0238] Figure 83. Leaves from A. white clover, B. canola and C. wheat plants exhibiting delayed leaf senescence (levels from LXR® transgenic plants, lower images) when compared to control plants negative (leaves from control plants, top images) 7-20 days following detachment of leaves from plants.

[0239] Figure 84. Positive selection of transgenic perennial ryegrass plants by selection of phenotypes grown in vitro on plates without antibiotic selection. B.C. Calluses in the dark for 8 weeks after transformation; D-F. 1 week after transfer to the light.

[0240] Figure 85. Embryonic perennial ryegrass calli bombarded with gold particles alone (control) and with gold particles coated with FTTaRbcS fusion vector before, and four weeks after, transfer to light.

[0241] Figure 86. Embryonic perennial ryegrass callus bombarded only with gold particles (control) and with gold particles coated only with FT TaRbcS fusion vector 1, only with FT TaRbcS fusion vector 3, only with FT TaRbcS vector LXR®, as well as with FT TaRbcS plus LXR® fusion vectors, five weeks after transfer to light. Molecular analysis positive lines: FT TaRbcS FT fusion 1 #1, 2, 3, 4, 7, 6, 12, 13, 14, 16, 17; FT TaRbcS fusion 3 #1, 2, 3, 5, 8,10,11,12,13; FTTaRbcS fusion 1 plus LXR® #1, 2, 7,12 (FT TaRbcS fusion only); #8, 14 (fusion 1 of FTTaRbcS plus LXR®).

Example 1 Isolation of photosynthetic promoters Cloning of a photosynthetic promoter from bread wheat

[0242] The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) is a well-characterized light-regulated gene in higher plants. From bread wheat (Triticum aestivum), TaRbcS regulatory sequences (promoter and terminator) were previously cloned (Zeng, et al., 1995; Sasanuma, 2001). A 695 bp promoter fragment from a previously published sequence containing the signal TATA from the TaRbcS gene (accession number NCBIAB042069) was amplified by PCR.

Cloning of a photosynthetic promoter from Arabidopsis

[0243] A 1,700 bp fragment of the Arabidopsis thaiiana ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit promoter sequence (AtRbcS) had previously been cloned. Primers will be designed to amplify a smaller fragment containing the TATA signal from the AtRbcS promoter for use in expression vectors.

Discovery and cloning of photosynthetic promoters from perennial ryegrass

[0244] The expression of RbcS and chlorophyll a/b binding protein (CAB) are well-characterized light-regulated genes in higher plants. The abundance of LpRbcS mRNA transcripts in perennial ryegrass by quantitative real-time PCR is illustrated in Figure 2.

[0245] Both the LpRbcS genes and the LpCAB genes were chosen for promoter discovery and isolation in perennial ryegrass. Publicly available cDNA sequences (LpRbcS, EC778430 and LpCAB, EC778438) were used as query sequences in a BLAST search of the perennial ryegrass EST database in our own domestic database. As both genes are members of multigene families, several contigs (each contig represents an individual gene) were identified in our perennial ryegrass EST collection. Nine contigs were found to be homologous to the published LpRbcS cDNA sequence and thirteen contigs were found to be to the LpCAB cDNA sequence. Two contigs, LPCL9_C359 (LpRbcS) and LpCL1112_C12 (LpCAB), representing the promoter genes to be isolated, contained (47) and (19) EST sequences, respectively. These sequences came from a variety of libraries representing a range of different tissues. These data were used for in siiico expression analysis and indicated that both genes are only expressed in photosynthetic tissues (Figure 3).

[0246] DNA sequence alignments for each of the gene family members were performed, and gene-specific primers were designed for contigs LpRbcS_C359 and LpCAB_C12 and used to select groups of perennial ryegrass BAC DNA by PCR. BAC clones were identified and sequenced. Primers were designed and promoter regulatory sequences specific to LoHum perenne were cloned, sequenced (Figures 4 and 5) and cis-regulatory sequences specific to photosynthetic promoters were identified PLACE (http://www.dna.affrc.go.jp /PLACE/) (Table 1). The sequences included the I-Box motif to GT1 box for RbcS (Terzaghi, et al., 1995; Martinez-Hernandez, et al., 2002). In addition, 16/19 nucleotides of the LpRbcS sequence shared homology with the monocot RbcS consensus sequence (Schaffner, et al., 1991). The I-Core box and SORLIPs cis-regulatory sequences were present in the CAB promoter. SORLIPs were found to be overrepresented at light-regulated promoters in Arabidopsis (Hudson, et al., 2003). Table 1. The position of cis-regulatory sequences identified by the PLACE database. Common cis-acting regulatory sequences are listed. (Schaffner, et al., 1991; Terzaghi, et al., 1995; Martinez-Hernandez, et al., 2002; Hudson, et al., 2003). The positions observed are the first nucleotide in the sequence, relative to the ATG (np - not present).

[0247] These L. perenne-specific promoter regulatory sequences were subsequently used in the construction of backbone-free expression cassettes with fructan biosynthesis genes.

Example 2 Isolation of fructan biosynthesis genes Isolation of fructan biosynthesis genes from LoHum perenne

[0248] Perennial LoHum cDNA clones encoding sequences for Lpl-SST and Lp6G-FFT were previously isolated from a perennial ryegrass cDNA library (Chalmers, etaL, 2003; Chalmers, etaL, 2005). The complete gene sequences of the isolated perennial ryegrass fructosyl transferase homologues are available, and nucleotide and protein sequences for Lpl-SST are disclosed in international patent application number PCT AU01/00705 (SEQ ID NOS lie 12).

[0249] The partial sequence for Lp6G-FFT is disclosed in international application number PCT/AU/01/01275 (SEQ ID NOS 109 and 110), for nucleotide and amino acid sequences, respectively. The full-length clone was amplified by PCR from cDNA, cloned and sequenced (Figure 7). When the Lp6G-FFT ORF was compared with the Lp6G-FFT published from L. perenne, 23 nucleotide changes were observed. Comparison of the predicted protein sequences revealed only two changes between the two amino acid sequences (Figure 8).

[0250] Other FT genes, which can be used and either transformed solely or cotransformed with Lpl-SST and Lp6G-FFT, include Lpl-FFT, Lp6-SFT and Lp6-SST. The cDNA sequence for Lpl-FFT was isolated from perennial ryegrass (Figure 9) and the amino acid sequence is depicted in Figure 10. As an example, primers based on this sequence could be used to amplify the full-length cDNA by PCR , for cloning and use in the present invention, as described below.

[0251] Other homologous proteins can be found by selection databases such as EMBL (httD://www.ebi.ac.uk/Tools/index.htm) or the National Center for Biotechnology Information (NCBI, http: //www.ncbi.nlm.nih.gov/blast/Blast.cgitf). In such a database search, for example, the sequences described in Figures 7-10 are fitted to a query, using default parameter settings tuned for the database. For example, for protein sequence alignments (Blastp) with NCBI, these settings are as follows: limit entrez=not activated; fiiter=iow complexity activated; expect vaiue=10; word size=3; matrix=BLOSUM; gapcostsexistence-11 ,extension=l. Such database searches can be used to find proteins with domains contained in TFs (using default parameters).

Example 3 Creation of translational TF fusion proteins FT translational FT fusion cloning

[0252] A genetic FT fusion was created between the open reading frames for Lpl-SST and Lp6G-FFT, following the procedure depicted in Figure 11. The Lpl-SST gene was amplified by PCR with an incorporated GATEWAY recombination site in the forward launcher. A sequence, which encodes three glycine residues followed by a HindIII site, was incorporated into the reverse primer, with the stop codon removed. The Lp6G-FFT gene was amplified by PCR with a Hind III site followed by a sequence encoding three glycine residues and gene-specific sequences without the ATG. The reverse primer for the Lp6G-FFT gene was flanked by a second GATEWAY recombination site. Primer sequences are provided in Table 2. Purified fragments were digested with Hind III and the ligated product was cloned into the GATEWAY vector pDONR221 Entry. When the resulting pENTRYl-Lpl-SST-Lp6G-FFT-2 entry clones were sequenced, one sequence (FT fusion 1) was confirmed to be the predicted product, with eight amino acids in the linker joining the two genes (Figures 12 and 13). . Whereas another sequence (TF fusion 3) contained two consecutive Hind III sites, which would result in the addition of two other amino acids, giving a total of ten amino acids between the two FT genes during translation (Figures 14 and 15). Table 2. Primer sequences used to amplify the PCR fragments used to generate the translational FT fusion of the Lpl-SST and Lp6G-FFT fructosyl transferase genes (Lpl-SST_Lp6G-FFT of FT). Shaded sequences are gene specific, bold and underlined sequences (ER Hind III site) are nucleotides introduced to generate the linker region, and italicized nucleotides represent recombination-specific sequences.

[0253] By use of the primer sequences shown in Table 3, it is possible to create a new FT fusion by reversing the order to Lp6G-FFT_Lp 1-SST using the same method as illustrated above. Table 3. Primer sequences used to amplify the PCR fragments used to generate the translational FT fusion of the fructosyl transferase genes LplSST and Lp6G-FFT (Lp6G-FFT_Lpl-SST). Shaded sequences are gene specific, bold and underlined sequences (ER Hind III site) are nucleotides introduced to generate the linker region, and italicized nucleotides represent recombination-specific sequences.

[0254] In Lpl-SST_Lp6G-FFT, the FT proteins physically associate with each other to form a FT fusion protein, which contains three transmembrane domains as designed by SOSUI, a classification and prediction database of secondary structure of membrane proteins (Table 4, Figures 17 and 18). Table 4. FT 1/3 fusion transmembrane domains as indicated by SOSUI, a membrane protein secondary structure classification and prediction database (httD://bD.nuaD.nagova-u.ac.iD /sosui/sosui submit.html)

Structural features of plant fructosyl transferases

[0255] Plant TFs have a high degree of amino acid homology relative to vacuolar invertases (β-fructose), which are members of glycoside hydrolase family 32 (GH32) and share three highly conserved regions characterized by the motifs (N/ S)DPNG (also called β-fructosidase motif), RDP and EC (Altenbach et a!., 2005) (Figures 17, 18 and 36). Another common feature of plant TFs and vacuolar invertases is that they are usually composed of a large subunit and a small subunit due to post-translational processing. The large subunit, which harbors all three conserved motifs mentioned above, determines catalytic specificity (Altenbach eta/., 2004).

[0256] The other FT genes Lpl-FFT, Lp6-SFT and Lp6-SST, can also be used in combination with Lpl-SST or Lp6G-FFT, to produce a selection of translational FT fusions, by the scheme shown in Figure 16A, as indicated below. • Lp6G-FFT:: Lpl-SST • Lpl-SST: :(Lpl-FFT/Lp6-SFT/Lp-SST) • (Lpl-FFT/Lp6-SFT/Lp-SST):: Lpl-SST

[0257] A triplicate FT fusion could also be created using a similar methodology (Figure 16B). It is proposed that the triplicate fusion would be constructed to incorporate the Lpl-SST, Lp6G-FFT and Lpl-FFT, Lp6-SFT or Lp6-SST genes. By altering the primer sequences used to join the two FT genes together, it is possible to change the linker size and potentially add up to approximately 30 amino acids. FT proteins could physically associate with each other to form a metabolic channel, therefore, the distance separating FT genes within the translational fusion could affect protein function. FT fusion proteins preferably contain sequences representing domains that are highly conserved among FT, INV and FEH proteins from LoHum perenne plants indicated in Figures 17, 18 and 36.

Example 4 Transient function assays of fructan biosynthesis genes Function of Lpl-SST, Lp6G-FFT and FT fusion protein

[0258] The cDNA sequence, which encodes the mature Lpl-SST protein, was previously expressed in Pichia pastoris for functional characterization (Chalmers, et al., 2003) and the conversion of sucrose into 1-ketose, by expression of this protein, was demonstrated. Similarly, the Lp6G-FFT cDNA was cloned into the pPICZoA expression vector (Invitrogen), which contains a methanol-inducible promoter and the Saccharomyces cerevisiae o-factor sequence to allow secretion of the recombinant protein for isolation for functional characterization. The recombinant Lp6G-FFT enzyme was produced from single colonic transformed P. pastoris inoculated into a pre-culture medium and induced by the addition of methanol for 45 hours duration. The supernatant was concentrated and samples were incubated with 10 mM sucrose overnight. The carbohydrates produced were analyzed by CTAAD according to Chalmers, et al., 2003, using fructan extracts from onion as a control (Figure 19).

Generation of vectors for transient gene expression assays

[0259] Numerous vectors have been constructed using Invitrogen Multisite Gateway™ technology (see www.lnvitroqen.com for product manual) based on recombinant cloning. This methodology uses the regeneration of individual Entry plasmids containing each of the promoter, gene of interest (GOI) or terminator sequences flanked by recombination sites. The recombination sites facilitate triple-directional insertion of each of the Entry plasmids into a gate-enabled destination vector, by recombination. The final vector is then sequenced and used directly for cotransformation of plants with a plasmid, or expression cassette, for expression of a plant selectable marker.

[0260] In order to test the function of the FT fusion protein, FT fusion ORFs 1 and 3 were cloned under the control of the enhanced cauliflower mosaic virus (CAMV)35S2 promoter (Kay, et al. , 1987), using the Multisite Gateway™ technology recombination system (see www.lnvitrogen.com for product manual) in Agrobacterium binary vector (Figure 21) (Hajdukiewicz, et al., 1994).

[0261] Gate Entry vectors were constructed for the (CAMV)35S2 promoter, the TaRbcS terminator sequence, as well as FT fusion 1 and 3 ORFs. The cloned fragments were sequence checked and used for cloning by trilateral recombination with the GOI cDNA sequences cloned into the destination vector pPZP200-ubi:bar-nos R4 R3. In addition, the constructs also included the unique Lp6G-FFT and Lpl-SST ORF driven by the (CAMV)35S2 promoter as controls. As an example, the single ORF of Lpl-FFT (or Lp6-SFT, Lp6-SST) is also cloned in the same way. As a control, GUS ORF was used for expression confirmation. The following constructs were made. • pPZP200-35S2::Lp6G-FFT::TaRbcS • pPZP200-35S2::Lpl -SST::TaRbcS • pPZP200-35S2: :(Lpl-FFT/ Lp6-SFT/Lp-SST)::TaRbcS . pPZP200-35S2::Lpl-SST: :6G-FFT::TaRbcS (FT fusions 1 and 3) . pPZP200-35S2::GUS: TaRbcS

[0262] Using Invitrogen Multisite Gateway™ technology, the following vectors are created containing the Atrbcs photosynthetic promoter and the (CAMV)35S terminator for use in transient assays. • pPZP200-AtrbcS::Lpl-SST::35S . pPZP200-AtrbcS::Lp6G-FFT::35S • pPZP200-AtrbcS: :(Lpl-FFT/Lp6-SFT/Lp-SST): :35S • pPZP200-AtrbcS::Lpl-SST::6G-FFT::35S (FT mergers 1 and 3)

Function of Lpl-SST, Lp6G-FFT and FT fusion protein in transient transgene expression assays

[0263] For proof of function, transient expression of the constructs, containing genes for Lpl-SST, Lp6G-FFT and chimeric FT fusion proteins driven by the (CaMV)35S promoter, was conducted in tobacco plants, as they do not naturally produce fructans. The method involved agro-infiltration of the individual constructs into leaves of N. benthamiana (Kapila, et al., 1997; Wydro, et al., 2006) followed by biochemical analysis by anion exchange chromatography. A diagram of the transient expression procedure is illustrated in Figure 21. Three days after injection, the material was harvested and water-soluble carbohydrates using high-performance anion exchange chromatography (CTTAD). The results show production of fructans, with increased production of both 1-ketose and 6G-ketose by the FT fusion protein (Figure 22). An equivalent experiment is used to evaluate function constructs containing chimeric Lpl-SST, Lp6G-FFT, and FT fusion protein genes driven by the AtRbcS promoter.

Agro-infiltration using a combination of vectors for transcriptional cotransformation

[0264] To evaluate the function of fructan biosynthesis, when transcriptionally coordinated together in a cell, trilateral agro-infiltration experiments are conducted using the groups of vectors outlined below. The transient expression procedure as illustrated in Figure 21 is used to insert three vectors together into the same plant tissue. Three days after injection, the plant material is collected and the water-soluble carbohydrates are extracted using a hot water extraction method. Extracts are separated using high performance anion exchange chromatography (CTAAD). The results indicate differences resulting from the independent expression of three fructan biosynthesis genes in the plant genome. • pPZP200-35S2::Lp6G-FFT::TaRbcS + . pPZP200-35 S2::Lpl-SST:TaRbcS + • pPZP200-35 S2::(Lpl-FFT/Lp6-SFT/Lp-SST)::TaRbcS • pPZP200-AtRbcS::Lpl-SST::35S+ • pPZP200- AtRbcS:: Lp6G-FFT::35S + • pPZP200-AtRbcS: :(Lpl-FFT/Lp6-SFT/Lp-SST):: 35S

Agro-infiltration using FT fusion vectors for translational cotransformation

[0265] By comparison with transcriptional cotransformation, experiments are conducted to compare translational cotransformation, conducting transient assays with the vectors that had been previously discussed and which are indicated below. • pPZP200-35S2: :Lpl-SST_6G-FFT: : TaRbcS (FT fusions 1 and 3) • pPZP200-AtRbcS::Lpl-SST_6G-FFT::35S (FT fusions 1 and 3)

Example 5 Generation of vectors for stable transformation and production of transgenic plants Production of LXR® vector for biolistic and Agrobacteríum-mediated transformation

[0266] LXR® technology is based on vectors containing a cytokinin biosynthesis gene, which encodes isopentenyl transferase (IPT) for delayed leaf senescence, under the control of the AtMYB32 gene promoter. The LXR® vector, for biolistic transformation, was constructed using Gateway Multisite™ technology. Details of the binary vector pBS-ubi::bar::nos_AtMYB32_IPT_35S are described in international patent application number PCT/AUOl/01092.

[0267] The production of LXR® vectors for Agrobacteríum-mediated transformation is disclosed in international patent application number PCT/AUOl/01092. Candidate genetic constructs were completely sequenced and vectors were generated for Agrobacteríum-mediated transformation following strict quality assurance protocols.

Constructs containing a wheat photosynthetic promoter

[0268] A 695 Kb promoter fragment, from a previously published sequence containing the TATA signal from the TaRbcS gene (NCBI accession number AB042069) was amplified by PCR with Gateway™ recombination sites (Invitrogen) on the flanks of the launcher. The fragment was cloned into the Invitrogen pDONRP4-Pl R Entry vector using Gateway™ recombination technology. The 696-bp TaRbcS gene termination signal sequence (Sasanuma, 2001) was also amplified by PCR, using primers with recombination sites, and cloned into the Invitrogen pDONRP2-P3R Entry vector. The cloned fragments were sequence checked and used for cloning by trilateral recombination with the GOI cDNA sequences in the destination vector pDEST-R4R3: pDESTRl-R2R-Lpl-SST, pDESTRl-R2-Lp6G-FFT and pDESTPl-P2R- Lpl-SST_Lp6G-FFT, gene FT fusion expression vectors. The following constructs, for photosynthetic regulation of fructosyl transferase expression by the TaRbcS promoter to be used, are shown below and graphically depicted in Figure 23. Expression cassette sequences for FT fusions 1 and 3 pDEST-TaRbcS: :Lpl- SST ::TaRbcS, pDEST-TaRbcS::Lp6G-FFT::TaRbcS and pDEST-TaRbcS::Lpl-SST_Lp6G- FFT::TaRbcS are provided in Figures 24 - 27. • pDEST-TaRbcS: :Lpl-SST::TaRbcS • pDEST-TaRbcS:: Lp6G-FFT: :TaRbcS • FT merges 1 and 3 pDEST-TaRbcS: :Lpl-SST_Lp6G-FFT::TaRbcS . pDEST-TaRbcS: :GUS::TaRbcS

[0269] Constructs containing a ryegrass photosynthetic promoter were produced by conventional cloning methods. The 693 base pair (bp) fructosyltransferase 4 (LpFT4) gene termination sequence (Lidgett, et al., 2002) was amplified by PCR using gene-specific primers containing the restriction endonuclease site ( ER) of EcoR I at the 5' end of the forward PCR primer. EcoR 1/ and Xma I restriction endonuclease sites were incorporated into the 3' end of the reverse PCR primer. The PCR product was cloned into the EcoR I and

[0270] The LpRbcS promoter was amplified using gene-specific primers containing the Xho l and EcoR V restriction endonuclease sites at the 5' end of the forward primer, and an EcoR 7 restriction site was incorporated into the 3' end of the primer reverse. The 610-bp PCR product was cloned into the EcoRIe-digested plasmid pBS-LpFT4. Xho I, resulting in the plasmid pBS-LpRbcS::LpFT4 (Figure 29A). The Lpl-SST coding region was amplified from a cDNA template (Chalmers et al., 2003) with EcoR/Z sites flanking both forward and reverse PCR primers, and cloned into the EcoR Z site of the vector pBS- LpRbcS::LpFT4, generating the final construct pBS-LpRbcS::Lpl-SST::LpFT4 (Figure 29B). Sequence of the expression cassette, indicating promoter and terminator, as well as ORF, is provided in Figure 31. The expression cassette containing the L. perenne sequences can be excised from the plasmid vector DNA using restriction endonuclease EcoR k Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix before being used for plant transformation, to produce DNA without vector backbone sequences.

[0271] Plasmid pBS-LpFT4 (Figure 28), containing the 693 base pair LpFT4 terminator sequence, was prepared as outlined above. The 870 base pair LpCAB promoter fragment was amplified with a forward PCR primer containing the Xho Ze EcoR K sites and a reverse PCR primer containing the EcoR I restriction sites. Ze EcoR Ze of pBS-LpFT4, generating the plasmid pBS-LpCAB::LpFT4 (Figure 30A). The region encoding Lp6G-FFT was amplified from a cDNA template (Chalmers, et al., 2005) with EcoR I sites flanking both forward and reverse PCR primers, and cloned into the EcoR Z site of the pBS vector. -LpCAB::LpFT4, generating the final construct pBS- LpCAB::Lp6G-FFT::LpFT4 (Figure 30B). The sequence of the expression cassette, indicating promoter and terminator, as well as ORF, is provided in Figure 32. The DNA expression cassette can be excised from the plasmid vector DNA using EcoR k restriction endonuclease Following gel electrophoresis agarose matrix, the resulting DNA fragment is purified from the agarose matrix before being used for plant transformation, to produce DNA without vector backbone sequences.

[0272] To generate an expression construct, the translational FT fusion between the Lpl-SST and Lp6G-FFT genes was amplified from FT fusion 1 and 3 plasmids pDEST-TaRbcS::Lpl-SST_Lp6G-FFT:: TaRbcS FT (Figure 23C-D) using primers specific to a region just outside the ORF, with EcoR I restriction sites engineered into the 3' region in both the forward and reverse primers. The 3,920 bp ORF was amplified by PCR and cloned into the pCR®-Blunt vector (Invitrogen) to produce Blunt-Lpl-SST-Lp6G-FFT TF fusion by PCR (Figure 33). It was then excised using EcoR I restriction enzymes to remove the vector-specific sequences, and cloned into the plasmid pBS-LpRbcS::LpFT4 (Figure 29A) at the EcoR I restriction site, generating pBS-LpRbcS::Lpl -SST_Lp6G- FFT::LpFT4 (Figure 34). The sequence of the expression cassette of FT fusions 1 and 3 indicating relevant domains (FT fusion 3), is provided in Figure 35 and 36, respectively. The DNA expression cassette can be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix before being used for plant transformation, to produce DNA without vector backbone sequences.

[0273] The constructs for photosynthetic regulation of fructosyl transferase expression by L perenne promoter sequences are shown below: • pBS-LpRbcS:: Lpl-SST: :LpFT4 • pBS-LpCAB::Lp6G-FFT::LpFT4 • FT fusions 1 and 3 pBS-LpRbcS: :Lpl-SST_Lp6G-FFT::LpFT4

Constructs containing an Arabidopsis photosynthetic promoter

[0274] A construct containing an Arabidopsis photosynthetic promoter, compelling expression of FT fusion 3, was produced using Multisite Gateway cloning methods FT fusion 3 -pPZP200_AtRbcS::Lpl-SST_6G-FFT::35S (Figure 37). The sequence of the FT AtRbcS::Lpl-SST_6G-FFT::nos fusion 3 expression cassette is provided in Figure 38.

Construct containing a constitutive ubiquitin promoter

[0275] Constructs containing the promoter and first intron of the maize (Zea mays) ubiquitin (Ubi) gene (Christensen, et al., 1992) were produced by conventional cloning methods. The Ubi promoter is considered a constitutive promoter , but expression is highest in young tissues of actively growing grasses (Rooke, et al., 2000).

[0276] A cDNA copy of the candidate genes, Lpl-SST and Lp6G-FFT, was amplified by PCR as described by Chalmers, et al. (2003), and cloned into the pBlueScript SK(-) vector (Figure 39). The cDNA fragments were excised using the restriction endonucleases Xho Z and Xba I, and then cloned at the blunt end into the BamHZ site of the p-Ubi-35S vector (Figure 40). The binary plant expression vector p-Ubi-35S had been previously described in other L. multiflorum transformation experiments (Ye, et al., 2001). The clones p-Ubi::Lpl-SST: :35S and p-Ubi ::Lp6G-FFT::35S containing the DNA insertion in the required 5' to 3' orientation were confirmed by DNA sequencing. A representative sequence of the promoter (Ubi) constitutive with an FT fusion protein and a terminator sequence is provided in Figure 41. A similar method is used to construct p-Ubi::Lpl-FFT::35S clones.

[0277] The constructs for photosynthetic regulation of fructosyltransferase expression by Ubi promoter sequences are shown below. • p-Ubi: :Lpl-SST::35S • p-Ubi: :Lp6G-FFT::35S • p-Ubi:: (Lpl-FFT/Lp6-SFT/Lp-SST):: 35S

Constructs containing the cauliflower mosaic virus 35S promoter

[0278] Constructs for regulating expression of fructosyl transferases under the control of the enhanced cauliflower mosaic virus (CAMV)35S2 promoter (Kay, et al., 1987), are described in a previous section and are shown below. • pPZP200-35S2::Lp6G-FFT: :Ta RbcS • pPZP200-35S2:: Lpl-SST: :TaRbcS • pPZP200-35S2: :(Lpl-FFT/Lp6-SFT/Lp-SST): :TaRbcS • fusions 1 and 3 of FT pPZP200-35S2: :Lpl-SST_6G-FFT: :TaRbcS

Constructs containing tissue-specific or regulated promoters

[0279] Promoters with tissue specificity are desirable to compel expression of transgenes in cultures to direct accumulation in particular tissues/organs and to avoid unwanted expression elsewhere. Examples of different promoters, to compel expression of transgenes for different purposes, are presented in Table 5. Representative examples of constitutive (Ubi, (CAMV)35S2, RUBQ2, OsActl), tuber and runner specific (Cathlnh), stress-regulated promoters (Atrd29a) and sucrose-responsive (14-3-3 family of 16R proteins) linked to TF fusions are shown in Figures 42 - 48, respectively. Table 5. Examples of different promoters to compel transgene expression

[0280] Several photosynthetic promoters have been shown to be strong regulators of transgene expression in light-responsive tissues. The advantages of photosynthetic promoters for expression of fructan biosynthesis genes include the fact that they are active in the large group of cells in leaves and upper parts of stems, which comprise the majority of plant biomass. They are not expressed constitutively, however, their expression pattern overlaps, temporally and spatially, with sucrose accumulation.

Using a combination of vectors for translational cotransformation

[0281] The following vectors are transformed singly or in groups (double or triple) to evaluate the synergistic co-expression responses necessary for the generation of low and high GP fructans. • pDEST-TaRbcS::Lpl-SST::TaRbcS • pBS-LpRbcS:: Lpl-SST: :LpFT4 • p-Ubi::Lpl-SST::35S • pPZP200-35S2:: Lpl-SST: :TaRbcS • pDEST -TaRbcS:: Lp6G-FFT: :TaRbcS • pBS-LpCAB::Lp6G-FFT::LpFT4 • p-Ubi: :Lp6G-FFT::35S • pPZP200-35S2::Lp6G-FFT::TaRbcS • pDEST-TaRbcS : :(Lpl-FFT/Lp6-SFT/l_p-SST): :TaRbcS • p-Ubi:: (Lpl-FFT/Lpβ-SFT/Lp-SST):: 35S • pPZP200-35S2: :(Lpl-FFT /Lp6-SFT/Lp-SST): :TaRbcS

Using FT fusion vectors for translational cotransformation

[0282] To make comparisons with translational cotransformations, as indicated above, translational cotransformation experiments are also conducted with the FT fusion vectors, which have been previously discussed and are indicated below. • FT fusions 1 and 3 pDEST-TaRbcS: :Lpl-SST_Lp6G-FFT::TaRbcS • FT fusions 1 and 3 pBS-LpRbcS::Lpl-SST_Lp6G-FFT::LpFT4 • FT fusions 1 and 3 pPZP200-35S2 : :Lpl-SST_6G-FFT: : TaRbcS

Example 6 Production of stable transgenic plants by transformation Plant transformation

[0283] Genetic constructs of the present invention can be introduced into plant cells by transduction, transfection, transformation or gene targeting. Such techniques include Agrobacterium-mediated introduction, electroporation of tissues, cells and protoplasts, fusion of protoplasts, injection into reproductive organs, injection into immature embryos and introduction of high velocity projectiles into cells, tissues, callus immature and mature embryos, microinjection into cells and protoplasts, direct gene transfer mediated by polyethylene glycol, biolistic transformation, Whiskers transformation and combinations thereof. The choice of technique depends largely on the type of plant to be transformed and the appropriate vector for the chosen method is used.

[0284] Cells incorporating the genetic constructs of the present invention can be selected, as directed by the vectors used, and then cultured in an appropriate medium to regenerate transformed plants, using well-established techniques. The resulting plants can be reproduced, either sexually or asexually, to produce generations of transformed plants.

[0285] The present invention can be applied to a variety of plants, including monocotyledons [such as wheat, corn, rice, barley, sorghum, sugar cane, oats, rye, grasses (e.g., wheat, peat and grasses of bioenergy, including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, cane seed, big broom corn, cordgrass, napiergrass, switchgrass, wildrye, wild sugar cane, Miscanthus, Paspaium}}, dicots [ such as Arabidopsis, tobacco, soybeans, canola, alfalfa, cotton, potatoes, tomatoes, tobacco, clovers (e.g., white clover, red clover, underground clover), Brassica vegetables, lettuce, spinach] and gymnosperms. invention can be applied to cereals, such as Triticum aestivum (wheat), C3 grasses containing native fructans, such as LoHum perenne (ryegrass) and LoHum arundinaceum (tall fescue), as well as Paspaium diiatatum (paspaium} a perennial apomictic grass without fructans natives. The invention can also be applied to dicotyledons, such as Arabidopsis thaiiana, Brassica napus (canola), Nicotiana benthamiana (tobacco) and Trifoiium repe/?s (white clover).

Biological transformation of monocots, e.g. wheat, perennial ryegrass, tall fescue and paspaium

[0286] Candidate genes are inserted into the plant genome by particle bombardment using integral plasmids, thus main chain sequences can also be incorporated into the genome. Tissues from transgenic plants are coated for survival in tissue culture media containing a selective agent.

Agrobacterium-mediated transformation of dicots, e.g. Arabidopsis, tobacco, canola and white clover

[0287] Agrobacterium-mediated transformation takes advantage of the natural pathogenic activity of the soil bacterium Agrobacterium tumefaciens A. tumefaciens infects the roots and stems of dicotyledonous plants resulting in infection directed by the tumor-inducing plasmid (Ti) through the insertion of specific genes ( T-DNA) in the genome of infected plant cells. Candidate genes were inserted into the plant genome by Agrobacterium-mediated transformation using binary vectors based on Ti plasmids.

Example 7 Production of transgenic perennial grasses Use of constructs containing photosynthetic promoters

[0288] Biolistic cotransformation of perennial ryegrass with vectors containing the TaRbcS and LpRbcS regulatory sequences, compelling the expression of individual fructan genes or as a translational fusion of FT, and the pAcHl vector for hygromycin resistance has been previously constructed and has been used successfully in plant transformation experiments (Bilang, et a!., 1991; Spangenberg, et a!., 1995a; Spangenberg, et a!., 1995b; Ye, et a!., 1997; Bai, et al., 2001). The GUS marker gene was also cloned as a positive control. Table 6 summarizes the transformation and molecular analysis to generate these strains. Table 6. Summary of production of transgenic perennial ryegrass plants for expression of Lpl-SST and Lp6G-FFT and FT fusion ORFs, under control of photosynthetic promoter from wheat.

[0289] Cassette DNA containing L perenne sequences was excised from plasmid vectors pBS-LpRbcS::Lpl-SST::LpFT4, pBS-LpCAB::Lp6G-FFT::LpFT4 and pBS- LpRbcS::Lpl- SST_Lp6G-FFT::LpFT4 (Figures 29, 30 and 34, respectively) using the EcoR k restriction endonuclease Following agarose gel electrophoresis, the resulting DNA fragment was purified from the agarose gel before being used for transformation. of plants, to produce DNA without vector backbone sequences. The pAcHl vector, previously constructed and successfully used in plant transformation experiments, was also digested with restriction enzymes to produce a DNA fragment for expression of only the selectable marker.

[0290] The biolistic cotransformation of perennial ryegrass, with the vectors containing the L perenne regulatory sequences, compelling the expression of individual fructan genes or as a translational TF fusion, and the pAcHl expression cassette for hygromycin resistance, was conducted in embryonic callus for perennial ryegrass. Table 7 summarizes the transformation and molecular analysis to generate these strains. Table 7. Summary of transformation progress for production of transgenic perennial ryegrass plants for expression of Lpl-SST and Lp6G-FFT and FT fusion ORFs, under control of ryegrass photosynthetic promoters.

Example 8 Characterization of transgenic perennial grasses Characterization of transgenic FT and FT fusion perennial ryegrass plants

[0291] During the regeneration of transgenic perennial ryegrass plants, differences in growth phenotypes were observed between lines. Both tissue culture regenerants and plants grown in corresponding soil from both fusion 1 and fusion 3 transgenic plants showed a superior growth performance phenotype compared to transgenic plants containing either a single fructan biosynthesis gene or to control plants containing only the selectable marker, hph. Phenotypic examples of transgenic perennial ryegrass plants in tissue culture are shown for the TaRbcS promoter and LpRbcS FT fusion transgenics in Figures 48-51.

[0292] Plants showing the superior growth performance phenotype were conformed to contain the FT gene of interest. The superior growth performance phenotype of transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early stages of plant regeneration conducted on plates. Specifically, as early as 12 days after incubation under lights the transgenic calli showed additionally developed green shoots. The rapid growth rate of the FT fusion transgenic plants became more evident 22 days after transfer to rooting media. Transgenic plants containing either FT fusion 1 or FT fusion 3 constructs showed clearly increased numbers of shoots. In addition, FT fusion transgenic plants consistently showed a higher shoot density per plant compared to control plants in vitro (Figures 48-49).

[0293] Following transfer to soil and propagation under greenhouse conditions, more specific differences were observed between FT fusion 1 and FT fusion 3 strains. Although both FT fusion plants exhibited enhanced growth performance, FT fusion 1 plants had thicker, slightly darker green leaf blades. Also the plants were physically more robust with thicker leaf sheaths and leaf blades. The FT 3 fusion lines continued to grow faster than the other control plants with longer leaf blades and more vigorous shoot growth, but leaf morphology was more similar to the wild type. An increase in root biomass was also observed in transgenic perennial ryegrass plants grown in both FT fusion 1 and FT fusion 3 soil (Figure 50). Control transgenic plants harboring either Lpl-SST or Lp6G-FFT as single genes did not show the level of increased growth rate that was observed in the FT fusion 1 and 3 transgenic plants. Their appearances are similar to each other, although some developed more vigorously than transgenic plants containing either GUS or hph (Figure 50).

[0294] A phenotype similar to that observed in the greenhouse was also observed in the field. FT fusion transgenic plants showed a more vigorous growth phenotype with increased shoot number and longer leaf blades (Figure 51). Field test transgenic plants were analyzed for biomass production (Table 8). Biomass was evaluated, as shown in Figure 52, ranging from a rating of 1 having the lowest biomass to 5 having the highest. Table 8. Percentage of plants indicating range of biomass classifications by genotype observed under field test growth conditions.

[0295] Leaf blades from individual plants were cut and sectioned manually (Figure 53). Obvious differences seen were in the amount of chloroplasts in each cell, and in the number of cells with chloroplasts: being more in both transgenic TF fusion plants than in the control plants. In addition, chloroplasts were present in cells located on the abaxial side (bottom of the leaf) of transgenic plants, even though both plants were grown under the same light conditions in the growth environment. It was sometimes observed that control plants produced more chloroplasts in mesophyll cells located on the adaxial side (upper side facing the light source) than on the abaxial side, whereas transgenic plants very often produced almost equal numbers of chloroplasts on both sides.

[0296] Biochemical analysis by CTAAD of water-soluble carbohydrates, extracted from independent transformants harboring FT fusion 1 TaRbcS: :Lpl-SST_Lp6G- FFT::TaRbcS, FT fusion 3 TaRbcS::Lpl-SST_Lp6G-FFT:: TaRbcS, TaRbcS::Lpl- SST: TaRbcS, TaRbcS:: Lp6G-FFT::TaRbcS, and two control lines {hph only}, showed that FT fusion 1 and FT fusion 3 transgenic plants contained significantly higher levels high levels of total fructans (Figure 54), showing an increase of up to 2.5 times over control strains (Figure 54). In addition, 1-ketose levels were up to 4 times higher in FT 1 fusion lines (up to 3.7 μg/mg PS, total fructans: 20.5 μg/mg PS and sucrose 51.2 μg /mg PS), and 3-fold higher in FT fusion 3 lines (up to 2.4 μg/mg PS, total fructans: 26.0 μg/mg PS and sucrose 49.8 μg/mg PS ) compared to hph controls (Figure %%A-B). In TaRbcS::Lpl-SST:TaRbcS plants, 1-kestose increased up to 2.9 μg/mg PS (a 3-fold increase), whereas total fructans only increased 0.5-fold to 14 μg/mg from PS. In contrast, 1-ketose levels in the TaRbcS::Lp6G-FFT::TaRbcS transgenic plant lines showed marginal increases of up to 1.6 μg/mg PS for 1-ketose (up to 0.5-fold) and only one strain exhibited a small increase in total fructans for 10 μg/mg PS (Figures 55C-D and 56C-D). Analysis of the sucrose content of all lines revealed that some of the lines with high fructan content also exhibited an increase in total sucrose content (Figure 57).

[0297] Transgenic perennial ryegrass was also evaluated under field conditions for the level and composition of total fructans (Figures 58 and 59) and transgene expression (Figure 60C). Transgenic and control perennial ryegrass plants were sampled repeatedly throughout the field test growing season. Biochemical analysis of wild-type controls and independent transformants was conducted to show the level of total fructan per plant. Figure 58 illustrates fructan levels in milligrams (mg) per gram (g) dry weight (DS) of wild-type and transgenic field-grown whole shoots and leaf blades. Multiple individual FT fusion plants and LpRbcS::Lpl-SST transgenics were identified with fructan concentrations between 80 to 120 percent higher than wild-type (TS) control plants, in both shoot and blade samples. of leaves (Figure 58).

[0298] Representative results on the composition of fructans in leaf blades of three LpRbcS::Lpl-SST transgenic perennial ryegrass plants when compared to wild-type controls are shown in Figure 59. The results indicate an increased level of fructans from elevated GP in transgenic plants, which express LpRbcS::Lpl-SST (Box 1, Figure 59).

[0299] Transgene expression was detected in FT LpRbcS fusion and transgenic LpRbcS::LplSST perennial ryegrass plants analyzed by quantitative reverse transcription PCR (qRT-PCR) (Figure 60).

[0300] In order to quantify the increase in biomass single shoots were separated from each of the To transgenic lines and control lines, and propagated in mixture in pots under greenhouse conditions. After 7 weeks and 12 weeks, each plant was analyzed for plant height, leaf blade width and total number of shoots (Figures 61 and 62). After 7 weeks, control plants showed an average height of 24 cm, the average leaf width was 2.5 mm, and each plant had an average of two shoots. The transgenic FT fusion and fusion 3 lines, however, exhibited up to an 80% increase in plant height (43 cm), up to a 60% increase in leaf width (4 mm), and up to a 3-fold increase in number of shoots (6 shoots). After 12 weeks, control plants were on average 43 cm tall, leaf blade width was 3.5 mm, with 5 shoots per plant produced. During the same time period, transgenic FT fusion 1 and fusion 3 plants grew to 62 cm in height (43% increase compared to controls). Leaf width was up to 6 mm (70% increase) and the maximum number of shoots observed was 16 per plant (220% increase) (Figure 62).

Characterization of transgenic LXR® perennial ryegrass plants and fusion of FT plus transgenic LXR®

[0301] Cotransformation of the fusion of FT and LXR® technology produced an enhanced growth phenotype. Plants grown under greenhouse conditions exhibited increased shoot number and enhanced root biomass compared to control and transgenic LXR® plants alone (Figure 65).

[0302] Plant tissue dry weight experiments were conducted to establish the biomass of individual FT and LXR® fusion transgenic plants. Transgenic perennial ryegrass plants, grown under greenhouse conditions, were trimmed 5 mm below the lowest leaf sheath at the 10-shoot stage. After 6 weeks, all plant biomass from a height of 5 cm above ground level was collected in paper bags, oven-dried and weighed on a precision scale.

[0303] The control was calculated as the average of five independent gene-of-interest (GOI-ve) negative plants. Both FT fusion and FT plus LXR® fusion transgenic plants produced plants with a dry weight higher (up to two times) than the average level for the control (Figure 64).

[0304] Biochemical analysis of GOI-ve controls and independent transformants was also conducted to show total fructan levels per plant. Fructan levels in leaf blades of FT fusion transgenic plants alone, as well as FT plus LXR® fusion, exhibited an increase of up to sixfold compared to the average value of control plants (Figure 65).

Characterization of transgenic FT fusion tall fescue plants

[0305] Transformation of tall fescue grass was conducted with vectors containing the L. perenne regulatory sequences, compelling the translational fusion of FT, and the pAcHl expression cassette for hygromycin resistance. Transgenic tall fescue plants grown under greenhouse conditions exhibited increased shoot number and enhanced root biomass compared to control transgenic plants (Figure 66).

Characterization of transgenic LXR® tall fescue and transgenic FT plus LXR® fusion plants

[0306] Transgenic tall fescue plants {LoHum arundinaceum cv Jesup S3) that express only FT LpRbcS fusion 3, only FT TaRbcS fusion 3, as well as FT TaRbcS fusion 3 plus LXR® technology (AtMYB32::IPT) m set were produced. Table 9 summarizes the transformation and molecular analysis to generate these strains. Table 9. Summary of transformation progress for tall fescue with photosynthetically regulated expression of FT fusion 3 and/or LXR®.

[0307] Plant tissue dry weight experiments were conducted to establish the biomass of individual transgenic plants. Transgenic tall fescue plants, grown under greenhouse conditions, were trimmed 5 mm below the lowest leaf sheath at the 5-shoot stage. After 6 weeks, all plant biomass, from a height of 5 cm above ground level, was collected in paper bags, oven-dried and weighed on a precision scale.

[0308] The control was calculated as the average of five independent GOI-ve plants. Transgenic FT fusion tall fescue plants alone and transgenic FT plus LXR® fusion tall fescue plants both showed a two-fold increase in dry grass weight when compared to the average value of control plants (Figure 67).

[0309] Shoot number experiments were also conducted to establish the growth vigor of individual transgenic plants. Both transgenic tall fescue and GOI-ve control plants at the 5-shoot stage were trimmed as mentioned above and allowed to grow under greenhouse conditions for 6 weeks before shoot numbers were counted. The shoot number in the control represents the average shoot number obtained from five independent GOI-ve plants. Transgenic fusion lines of FT alone and FT plus LXR® fusion tall fescue plants showed up to a twofold increase in shoot number compared to the average value of control plants (Figure 68).

[0310] Transgenic tall fescue plants (5 shoots) were trimmed (as indicated above) and grown under greenhouse conditions for 6 weeks, when leaf blades were collected and freeze-dried for fructan analysis. The average fructan level in controls represents data obtained from five independent GOI-ve plants. Transgenic lines of FT fusion tall fescue plants show a dramatic increase (between three to fivefold) in fructan accumulation in leaf blades compared to the average fructan level in GOI-ve control plants (Figures 69).

Example 9 Production of transgenic wheat plants Transformation of single fructan genes expressing light-regulated promoter or the FT translational fusion

[0311] Biolistic transformation of wheat was conducted with vectors containing photosynthetic promoter regulatory sequences, compelling the expression of individual fructan genes or as a translational TF fusion, and a vector containing a selectable marker gene Ubi::bar:: chimeric nos for resistance to glysofinate (pAcH25), in wheat embryonic callus.

Transformation of AtMYB32 promoter and IPT gene for delayed senescence

[0312] A transformation vector was constructed for biolistic transformation of wheat containing the chimeric AtMYB32::IPT::35S with a chimeric Ubi::bar::nos selectable marker gene for glyphosinate resistance. The genetic transformation of wheat with the LXR® vector was based on biolistic transformation of embryonic callus from the wheat line Triticum aestivum L Bobwhite 26, as described in the international patent application PCT/AUOl/01092. The candidate gene was inserted into the wheat genome by particle bombardment using complete plasmids, so vector backbone sequences can also be incorporated into the genome. Transgenic plant tissues were recovered by survival in tissue culture media containing a selective agent.

Production of transgenic plants for reprogrammed fructan biosynthesis in photosynthetic cells and for extended life of photosynthetic cells

[0313] Using the methods shown above, transgenic plants were generated which contain both fructan biosynthetic genes driven by light-regulated promoters and LXR® technology genes for reprogrammed fructan biosynthesis in photosynthetic cells and for extended life of photosynthetic cells . Table 8 summarizes the transformation and molecular analysis for the generation of these transgenic plants. Table 10. Summary of transformation progress, for production of transgenic wheat plants for expression of Lpl-SST and Lp6G-FFT and FT fusion ORFs under control of wheat photosynthetic promoters and in combination with LXR® technology, for biosynthesis of reprogrammed fructan in photosynthetic cells and for extended life of photosynthetic cells.

Example 10 Characterization of transgenic wheat plants Characterization of transgenic FT fusion wheat plants

[0314] During the regeneration of transgenic wheat plants, differences in growth phenotypes were observed in vitro. Tissue culture regenerants from both FT fusion 1 and FT fusion 3 transgenic plants showed a superior vigorous phenotype compared to control plants.

[0315] The superior growth phenotype of transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early steps of in vitro plant regeneration, conducted in tissue culture plates. Following biolistic transformation, calli were maintained for two weeks in tissue culture dishes in the dark and then transferred to light conditions. Approximately 6 weeks after incubation under light conditions, the transformed calli showed more fully developed green shoots and the roots of the FT fusion transgenic regenerants grew at an extremely advanced rate (Figure 70).

[0316] The rapid growth rate of FT fusion transgenic plants became more evident after transfer to rooting media. FT fusion transgenic plants showed an obvious early increase in shoot number at around 2 months when compared to null controls (up to 5 shoots compared to one shoot observed in control plants). The leaf width of some of the plants was 4-5 mm compared to control plants 2-3 mm. In addition, FT fusion transgenics consistently showed a higher shoot density per plant compared to control lines (Figure 71).

[0317] Following transfer to soil and propagation under greenhouse conditions, transgenic wheat plants containing the FT fusion constructs will continue to show an increased number of shoots when compared to control plants ( Figure 72).

Characterization of transgenic LXR® and transgenic FT plus LXR® fusion wheat plants

[0318] Transgenic wheat plants, which contain the LXR® technology construct, showed an increased number of shoots when compared to control plants under greenhouse conditions (Figure 73A). They also showed an increase in photosynthetic tissue after 35 days under greenhouse conditions (Figure 73B).

[0319] Cotransformation of the FT fusion construct and LXR® technology produced an enhanced growth phenotype of greenhouse-grown plants. Some of the plants also showed obvious late senescence (at 40 days) under greenhouse conditions (Figure 74). Transgenic wheat plants expressing the FT fusion construct and the FT plus LXR® fusion construct also showed an enhanced level of fructans in leaves and an increased number of shoots when compared to control plants under greenhouse conditions ( Figure 75).

[0320] Biochemical analysis of GOI-ve controls, as well as independent FT fusion and FT plus LXR® fusion Ti wheat transformants, grown under greenhouse conditions, was conducted to show levels of total fructan per plant . A dramatic increase in fructan level (up to fivefold) was detected for both transgenic lines (Figure 76).

Example 11 Production of transgenic Paspa/um düatatum plants Transformation of the IPT gene under control of the AtMYB32 promoter for delayed leaf senescence

[0321] Genetic transformation of Paspalum dilatatum (apomictic dallisgrass) was based on biolistic transformation, as described in international patent application PCT/AUOl/01092.

[0322] The candidate gene expression construct was inserted into the Paspalum dilatatum genome by particle bombardment using complete plasmids, so vector backbone sequences can also be incorporated into the genome. Tissues from transgenic plants were recovered by survival in tissue culture media containing a selective agent.

Translational fusion transformation of FT under light-regulated promoter control for engineering fructan biosynthesis in photosynthetic cells

[0323] Genetic transformation of Paspalum dilatatum, with photosynthetic regulated fructan biosynthetic genes, is conducted using the same method that was used to produce the LXR® transgenic Paspalum dilatatum plants.

Example 12 Characterization of transgenic Paspalum dilatatum plants LXR® transgenic plants exhibit a superior growth phenotype

[0324] Transgenic Paspalum dilatatum plants, which express the IPT gene under control of the AtMYB32 promoter, revealed an enhanced biomass accumulation. During regeneration of putative transgenic P. dilatatum plants, differences in growth phenotypes were observed, showing a superior growth phenotype compared to control plants. The distinctive growth phenotype can be used as a selection tool for identifying transformed plants in combination with cotransformed vectors.

Example 13 Production of transgenic dicotyledonous plants Transformation of dicotyledonous plants from LXR® and fusion of FT plus LXR®

[0325] Binary vectors, containing the fusion of FT and LXR® technology, were generated for Agrobacteríum-mediated transformation of dicotyledonous plants. Transformation vectors also contained a chimeric 35S: :nptll: :35S or 35S: :hph: :35S as selectable marker genes.

Production of transgenic dicotyledonous plants

[0326] Transgenic white clover (Trifolium repens) and Arabidopsis thaliana plants, which express LXR® technology alone (AtMYB32::IPT), FT fusion AtRbcS: :Lpl-SST_Lp6G-FFT: :35S alone, as well as LXR® and the FT fusion AtRbcS::Lpl-SST_Lp6G-FFT::35S together were produced (Figures 77 and 80). Tables 11 and 12 summarize the transformation and molecular analysis for the generation of these strains, respectively. Table 11. Summary of transformation progress to white clover with photosynthetically regulated expression of FT and/or LXR® fusion Arabidopsis Table 12. Summary of transformation progress for Arabidopsis with photosynthetically regulated expression of FT and/or LXR® fusion Arabidopsis

Characterization of transgenic white clover plants

[0327] Quantitative RT-PCR was used to confirm transformants and to detect expression levels of the FT AtRbcS fusion in selected lines (Figure 78). These lines, exhibiting expression of the transgene, also demonstrated an increased level of fructans (Figure 68B). No expression was detected in control lines (Figure 78).

[0328] Biochemical analysis by CTAAD of water-soluble carbohydrates, extracted from independent transformants, expressing only AtRbcS FT fusion, AtRbcS FT fusion plus LXR® and GOI-ve control strains, was conducted to show total fructans levels per plant. FT AtRbcS fusion and FT AtRbcS plus LXR® fusion transgenic lines exhibited a twofold increase in fructan accumulation in leaves greater than that observed in controls (Figure 79).

Characterization of transgenic Arabidopsis plants

[0329] Quantitative RT-PCR was used to confirm transformants and to detect expression levels of the FT AtRbcS fusion in selected lines (Figure 81). Transgenic FT T2 fusion Arabidopsis plants, grown in soil, are shown in Figure 82. Gene of interest negative plants (GOI-ve) are also shown and do not show any phenotypic differences to transgenic FT fusion plants. express the transgene.

[0330] Binary vectors have also been used for Agrobacterium-mediated transformation of hypocotyledonous segments of Brassica napus (canola) (Patent PCT/AUOl/01092).

Example 14 Characterization of transgenic dicotyledonous plants Characterization of transgenic LXR® dicotyledonous plants

[0331] A functionally active fragment of the AtMYB32 promoter was used to compel expression of IPT in transgenic white clover and canola plants, as described in international patent application number PCT/AUO1/01092. The results observed from LXR® technology in dicotyledonous plants had been delayed leaf senescence; intensified leaf growth dynamics; reduced runner death; intensified biomass production; increased cumulative green leaf area; increased seed yield; increased aridity tolerance; increased shading tolerance; intensified pasture quality reflected by intensified ruminal fermentation kinetics and higher dry matter digestibility.

Transgenic plants exhibit a delayed leaf senescence phenotype

[0332] The regulation of developmental senescence can be assessed by simulating and initiating artificial aging of detached leaves in vitro on moist filter paper. Incubation of highlighted leaves in the dark is highly effective in inducing Senescence-Associated Genes (GASS), leaf yellowing and chlorophyll loss (Weaver and Amasino, 2001). Figure 83 demonstrates the highlighted senescence assay data associated with expression of the IPT gene, under the control of one of the two functionally active fragments of the AtMYB32 promoter, in white clover and canola. Transgenic plants exhibited a significant delay of leaf senescence when compared to leaves of control plants 7-20 days following detachment.

Example 15 Production of transgenic plants for reprogramming fructan biosynthesis in photosynthetic cells and for extended life of these photosynthetic cells

[0333] Using the methods shown above, transgenic plants were generated, which contain both fructan biosynthetic genes (FT including FT fusion genes), under the control of light-regulated photosynthetic promoters, for reprogramming fructan biosynthesis in photosynthetic cells and LXR® technology through co-expression of IPT gene driven by the AtMYB32 promoter to extend the life of photosynthetic cells.

Example 16 Use of distinctive growth phenotype as a selection tool to identify transgenic plants in vitro

[0334] The superior growth phenotype of transgenic FT fusion 1 or FT fusion 3 plants was observed in all types of plants from which it transformed (e.g., perennial ryegrass and wheat). In both ryegrass and wheat, it was first observed during the early stages of plate-driven plant regeneration. In experiments conducted without selection with antibiotics, the strong induction of shoots was observed at the stage when, after bombardment, the calli had been kept in dark conditions for 8 weeks. (Figure 84 A-C). After transferring the plates to light conditions (7 days after transfer), strong shoot induction was observed in the transgenic plants and much lower level of regeneration was detected in control plants (Figure 84 D-F).

[0335] Expression of the FT fusion, under control of TaRbcS promoters or other photosynthetic or constitutive sucrose-regulated promoters, could be used as a selection tool for identifying transformed plants at the tissue culture stage. Expression of the TF fusion protein can also be driven by a set of promoters, which are active due to the high concentration of sucrose that exists in the tissue culture medium, and much less active at the low levels of sucrose present in plants grown in the tissue culture. ground. The transgene can be subsequently secreted away from the transgenic plants in successive generations. The increased biomass of the transformed plants, to be used as the selective agent, should not require an antibiotic resistance marker for the selection process, allowing the production of a market-ready product.

[0336] The analysis was performed to evaluate the use of the distinctive growth phenotype in detecting a positive transformation outcome in perennial ryegrass. Calli of embryonic perennial ryegrass FLP410-20 were bombarded with gold particles coated only in FT TaRbcS fusion vector 1, only in FT TaRbcS fusion vector 3, only in AtMYB32::IPT (LXR®) vectors, as well as FT TaRbcS plus LXR® fusion vectors 1 without any selectable marker. Control calluses were bombarded with gold particles only.

[0337] Plants were regenerated without antibiotic selection and maintained for 2 weeks under dark conditions and then transferred to light conditions (16/8 hour light/dark photoperiod). Plant growth was examined before transfer to light and weekly for five weeks under light conditions. Calluses were maintained under nutrient deprivation conditions in a progressive manner on the same plate for five weeks (Callus induction medium: full strength SM + 250 mg/L L-asparagine + 2.5 mg/L 2,4- D + 6% sucrose + 0.7% agar).

[0338] Growth of control plants was initiated during the first two or three weeks under light conditions, but became significantly slower four and five weeks later (Figure 85). Some calli bombarded with FT TaRbcS fusion vectors showed more vigorous growth for two to three weeks and continued growth (at a reduced rate) in weeks four and five (Figure 85). No obvious differences were observed for calluses bombarded with LXR® alone.

[0339] Cotransformation with FT TaRbcS fusion vector 1 plus LXR® showed an intermediate phenotype between the control and FT TaRbcS fusion vector 1 alone (Figure 86).

[0340] Molecular analysis was undertaken to detect the presence of the FT TaRbcS fusion transgenes using qRT-PCR in putative transgenic lines. FT fusion transgenics exhibited between 60% and 70% transformation and selection efficiency without antibiotics. No single LXR transgenic plant showed the presence of the transgene. Fusion cotransformation of FtTaRbcS and LXR exhibited an 11% cotransformation and selection efficiency (Figure 86).

[0341] A co-transformation method of FT and LXR® fusions for positive selection, to determine co-transformation efficiency, had been developed and is shown below.

[0342] Initially, cotransformation efficiency is determined for a variety of transformation events, which include a vector containing an antibiotic selectable marker. These cotransformation events include: 1. FT fusion regulated by a photosynthetic promoter + selectable marker 2. LXR® plus hph selectable marker 3. FT fusion regulated by a photosynthetic promoter plus LXR® plus hph selectable marker

[0343] Selection occurs in antibiotic media for transgenics and the presence of the transgene for each double or triple cotransformation event is determined, generating a transformation efficiency number for each event.

[0344] A second round of cotransformation events also occurs without an antibiotic selectable marker in selection-free media. These cotransformation events include: 1. FT fusion regulated by a photosynthetic promoter + dsRED marker 2. LXR® plus dsRED marker 3. FT fusion regulated by a photosynthetic promoter plus LXR® plus dsRED marker

[0345] Selection for increased growth rate of shoots and/or roots occurs and the presence of the transgene from each double or triple cotransformation event is determined. The presence of the dsRED marker gene is easily determined by fluorescence visualization and helps determine the cotransformation efficiency for each of the transformation events. Comparison of cotransformation efficiencies, determined with and without a selectable marker, assists in establishing the effectiveness of using a superior phenotype as a selection tool.

References

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Claims (12)

1. Method for enhancing the productivity of a fructan biosynthetic pathway characterized by comprising the steps of: introducing into said photosynthetic cells of a plant of Lolium or Triticum species a genetic construct including a light-regulated promoter, operably binding to nucleic acids , which encode two fructan biosynthetic enzymes selected from the group consisting of: 1-SST wherein said nucleic acid encoding 1-SST includes a sequence selected from the group consisting of SEQ ID NO: 62, and the nucleotide sequences which encode the polypeptide sequence shown in SEQ ID NO: 63; 1-FFT wherein said nucleic acid encoding 1-FFT includes a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 64, SEQ ID NOS: 65-70, and the nucleotide sequences encoding the polypeptide sequences shown in SEQ ID NO: 6, SEQ ID NO: 71, and SEQ ID NO: 72; 6-SFT wherein said nucleic acid encoding 6-SFT includes a sequence selected from the group consisting of SEQ ID NOS: 73-75; and 6G-FFT wherein said nucleic acid encoding 6G-SFT includes a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 76, and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 4, and SEQ ID NO: 77; and said nucleic acids are linked to form a fusion gene encoding a fusion protein of the two enzymes. 2. Method according to claim 1, characterized in that the fusion gene includes a sequence selected from the group consisting of sequences presented in SEQ ID NOS: 7, 9 and 34-50, and the nucleic acid sequences encoding the sequences of polypeptides shown in SEQ. ID NOS: 8 and 10. 3. Method according to claim 2, characterized in that the fusion protein is encoded by a sequence selected from the group consisting of sequences presented in SEQ ID NOS: 7, 9 and 34-50, or includes a sequence as presented in SEQ ID NO: 8 or 10. 4. Method according to claim 1, characterized in that said two fructan biosynthetic enzymes are 1-SST and 6G-FFT. 5. Method according to claim 1, characterized in that said two fructan biosynthetic enzymes are 1-SST and 6-SFT. 6. Method according to claim 1, characterized by additionally including introducing into the plant a genetic construct capable of manipulating senescence in the plant, said genetic construct including a MYB gene promoter including a sequence shown in SEQ ID NO: 78 or a modified MYB gene promoter including a sequence selected from the group consisting of SEQ ID NOS: 79-81, operatively linked to a gene, encoding an enzyme involved in biosynthesis of a cytokinin wherein said gene encoding an enzyme involved in the biosynthesis of a cytokinin includes a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOS: 82-84 and sequences encoding the polypeptides shown in SEQ ID NOS: 85-87, or a variant thereof. 7. Genetic construct, as defined in claim 1, capable of manipulating fructan biosynthesis in photosynthetic cells of a plant of Tolium or Triticum species, the genetic construct characterized by including a light-regulated promoter, operatively linked to nucleic acids, which encode two fructan biosynthetic enzymes selected from the group consisting of - 1-SST wherein said nucleic acid encoding 1-SST includes a sequence selected from the group consisting of SEQ ID NO: 62, and the nucleotide sequences which encode the polypeptide sequence shown in SEQ ID NO: 63; - 1-FFT wherein said nucleic acid encoding 1-FFT includes a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 64, SEQ ID NOS: 65 and 66-70, and the nucleotide sequences which encode the polypeptide sequences shown in SEQ ID NO: 6, SEQ ID NO: 71, and SEQ ID NO: 72; - 6-SFT wherein said nucleic acid encoding 6-SFT includes a sequence selected from the group consisting of SEQ ID NOS: 73-75; and - 6G-FFT wherein said nucleic acid encoding 6G-SFT includes a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 76, and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 4, and SEQ ID NO: 77; and said nucleic acids are linked to form a fusion gene encoding a fusion protein of the two enzymes. 8. Genetic construct according to claim 7, characterized in that the two fructan biosynthetic enzymes are 1-SST and 6G-FFT. 9. Genetic construct according to claim 7, characterized in that the two fructan biosynthetic enzymes are 1-SST and 6-SFT. 10. Genetic construct as defined in claim 1, capable of intensifying the productivity of a fructan biochemical pathway in a plant, or of intensifying biomass in a plant of Tolium or Triticum species, wherein the genetic construct is characterized by including nucleic acids , which encode two enzymes selected from the group consisting of - 1-SST wherein said nucleic acid encoding 1-SST includes a sequence selected from the group consisting of SEQ ID NO: 62, and the nucleotide sequences encoding the polypeptide sequence shown in SEQ ID NO: 63; - 1-FFT wherein said nucleic acid encoding 1-FFT includes a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 64, SEQ ID NOS: 65 and 66-70, and the nucleotide sequences which encode the polypeptide sequences shown in SEQ ID NO: 6, SEQ ID NO: 71, and SEQ ID NO: 72; - 6-SFT wherein said nucleic acid encoding 6-SFT includes a sequence selected from the group consisting of SEQ ID NOS: 73-75; and - 6G-FFT wherein said nucleic acid encoding 6G-SFT includes a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 76, and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 4, and SEQ ID NO: 77; and the nucleic acids are linked to form a fusion gene, which encodes a fusion protein of the two enzymes. 11. Genetic construct according to claim 10, characterized in that the two fructan biosynthetic enzymes are 1-SST and 6G-FFT. 12. Genetic construct according to claim 10, characterized in that the two fructan biosynthetic enzymes are 1-SST and 6-SFT.