WO2014198885A1 - Fibres végétales à propriétés de coloration améliorées - Google Patents

Fibres végétales à propriétés de coloration améliorées Download PDF

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WO2014198885A1
WO2014198885A1 PCT/EP2014/062363 EP2014062363W WO2014198885A1 WO 2014198885 A1 WO2014198885 A1 WO 2014198885A1 EP 2014062363 W EP2014062363 W EP 2014062363W WO 2014198885 A1 WO2014198885 A1 WO 2014198885A1
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plant
cotton
promoter
fiber
xyloglucan
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PCT/EP2014/062363
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English (en)
Inventor
Frank Meulewaeter
Karel JANSSEUNE
Karen De Clerck
Lieve VAN LANDUYT
Gary Henniger
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Bayer Cropscience Nv
Universiteit Gent
Bayer Cropscience Lp
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Publication of WO2014198885A1 publication Critical patent/WO2014198885A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the current invention relates to altered xyloglucan levels in plant cell walls, preferably secondary plant cell walls, particularly secondary plant cell walls as they can be found in natural fibers or fiber producing plants.
  • the altered xyloglucan level leads to altered fiber properties.
  • fibers with increased xyloglucan levels have an increased capacity for uptake of dyes, including direct dyes, but have also an increased capacity for uptake of other substances such as e.g. flame-retardants, medical compounds, wrinkle-free agents or softeners.
  • Natural fibers including cellulose containing natural fibers from plants, such as cotton and linen, have been used by civilization for more than 5000 years. Natural cellulose containing fibers, however, do not possess the chemical versatility of synthetic fibers, due to the relative inert nature of the cellulose consisting of ⁇ -1-4 linked glucose monomers.
  • Direct dyes create a relatively weak hydrogen bond with the cellulose polymer forming a semi-permanent attachment. Direct dyes are easier to use and less expensive than fiber-reactive dyes, but do not withstand well washing. Fiber-reactive dyes are molecules that combine chromophores with a reactive group that forms strong covalent bonds with the fiber via reaction with hydroxyl groups. The covalent bonds provide a good resistance of the dyed fiber against laundring. Colorfastness can be improved using cationic fixatives. [05] During the dyeing process, large amounts of electrolytes are needed to shield the anionic dyes from the anionic fiber charges. Unreacted dyes (up to 40%) need to be removed by a washing step, generating large volumes of wastewater, also containing the above mentioned electrolytes.
  • WO2006/136351 provides methods and means for the modification of the reactivity of plant cell walls, particularly as they can be found in natural fibers of fiber producing plants by inclusion of positively charged oligosaccharides or polysaccharides into the cell wall. This can be conveniently achieved by expressing a chimeric gene encoding an N-acetylglucosamine transferase, particularly an N-acetylglucosamine transferase, capable of being targeted to the membranes of the Golgi apparatus in cells of a plant.
  • One of the application is increased dyeability.
  • WOl 1/089021 provides methods and means for the modification of the reactivity of plant secondary cell walls, particularly in cotton cell walls found in cotton fibers. This can be conveniently achieved by expressing a chimeric gene encoding a Saprolegnia monoica chitin synthase in cotton plants.
  • WO12/048807 provides alternative methods and means to produce positively charged oligosaccharides in the plant cell wall by introducing into said plant cell a Nodulation C protein fused to a heterologous Golgi signal anchor sequence.
  • the invention provides a method for producing plant fibers with altered dyeing properties comprising the steps of providing a first recombinant DNA molecule to at least one cell developing into a fiber of a fiber-producing plant, said recombinant DNA molecule comprising the following operably linked DNA molecules: i.
  • a plant-expressible promoter such as a fiber-selective or fiber- preferential promoter including a promoter from a fiber-specific ⁇ - tubulin gene, a promoter from a fiber-specific actin gene, a promoter from a fiber specific lipid transfer protein gene from cotton, a promoter from an expansin gene from cotton, or a promoter from a chitinase gene in cotton; a promoter from cotton from a glucanase gene, a promoter of the cotton FS18 gene, a promoter of the SCW-PRP gene from cotton, a promoter of the FB8-like gene from cotton, particularly a promoter having the nucleotide sequence of SEQ ID No.
  • nucleotide position 4208 to nucleotide position 5615 or having the nucleotide sequence of SEQ ID No. 5 from nucleotide position 75 to 1482; ii. a DNA region encoding a cellulose synthase-like C protein, such as a cellulose synthase-like C protein selected from CSLC4 from Arabidopsis thaliana, TmCSLC from Tropaeolum majus, CSLC4 from Hordeum vulgare including a cellulose synthase-like C protein comprising an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID No. 2, or with the amino acid sequence encoded by the nucleotide sequence of SEQ ID No.
  • a DNA region encoding a cellulose synthase-like C protein such as a cellulose synthase-like C protein selected from CSLC4 from Arabidopsis thaliana, TmCSLC from Tropaeolum majus, CSLC4 from Hor
  • DNA region encoding said cellulose synthase-like C protein comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID No. 1 or with the nucleotide sequence of SEQ ID No. 5 from nucleotide position 1485 to nucleotide position 3506 ; and iii. optionally, a DNA region involved in transcription termination and polyadenylation; and b. collecting fibers from said fiber-producing plant having altered dyeing properties.
  • the method comprises the further step of providing a second recombinant DNA molecule, wherein the second recombinant DNA molecule comprises the following operably linked DNA molecules:
  • a plant-expressible promoter such as a fiber-selective or fiber- preferential promoter including a promoter from a fiber-specific ⁇ - tubulin gene, a promoter from a fiber-specific actin gene, a promoter from a fiber specific lipid transfer protein gene from cotton, a promoter from an expansin gene from cotton, or a promoter from a chitinase gene in cotton; a promoter from cotton from a glucanase gene, a promoter of the cotton FS18 gene, a promoter of the SCW-PRP gene from cotton, a promoter of the FB8-like gene from cotton, particularly a promoter having the nucleotide sequence of SEQ ID No.
  • XXT xyloglucan 6-xylosyltransferase protein
  • DNA region encoding said xyloglucan 6- xylosyltransferase protein comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID No. 3 or with the nucleotide sequence of SEQ ID No. 5 from nucleotide position 5618 to nucleotide position 7000; and iii. optionally, a DNA region involved in transcription termination and polyadenylation.
  • the fiber producing plant is selected from cotton, hemp or flax, preferably cotton.
  • the fibers may have an increased dyeability for dyes such as direct dyes.
  • the invention provides a first recombinant gene and/or second recombinant gene as herein described, characterized in the first and second recombinant gene comprise a heterologous plant-expressible promoter, which may be a fiber-selective or fiber-preferential promoter.
  • a plant cell or plant such as a fiber- developing cell from cotton, hemp or flax comprising a first or first and second recombinant gene as herein described.
  • the invention also provides fibers, such a cotton fibers, obtainable through the methods herein described and fibers, such as cotton fibers, which can be obtained from the transgenic plants herein described.
  • the invention relates to fibers, such as cotton fibers comprising an increased level of xyloglucan compared to fibers from unmodified plant not comprising a first, or a first and second recombinant construct as herein described.
  • fibers such as cotton fibers, comprising a xylose level, preferably terminal xylose, of more than 1% after 2 hours of hydrolysis by trifiuoroacetic acid.
  • Yet another objective of the invention is to provide yarns from the fibers herein described or fabrics from the yarns herein described.
  • the invention further describes the use of a first recombinant gene as herein described or of a first and second recombinant gene as herein described, to increase the dyeability of fibers harvested from fiber producing plants, preferably cotton or to increase the content of xyloglucan in fibers harvested from fiber producing plants, preferably cotton.
  • the invention further relates to the use of a DNA region encoding a cellulose synthase-like C protein or the combined use of a DNA region encoding a cellulose synthase-like C protein and a DNA region encoding a xyloglucan 6-xylosyltransferase to obtain plant cell walls, preferably secondary plant cell walls in fibers, with increased capacity for uptake of dyes, preferably direct dyes or to obtain plant cell walls, preferably secondary plant cell walls in fibers, with increased content of xyloglucan.
  • a method is provided to increase xyloglucan level in the plant cell wall, preferably the secondary cell wall of a plant comprising the step of providing a first and second recombinant DNA molecule to at least one cell of a plant,
  • the first recombinant DNA molecule comprises the following operably linked DNA molecules:
  • ii a DNA region encoding a cellulose synthase-like C protein; and iii. optionally, a DNA region involved in transcription termination and polyadenylation;
  • the second recombinant DNA molecule comprises the following operably linked DNA molecules:
  • XXT xyloglucan 6-xylosyltransferase protein
  • FIG. 1 Schematic representation of the results of the monosaccharide analysis of cotton fibers from transgenic cotton comprising recombinant CslC4 and XT1 constructs after trifluoroacetic acid mediated hydrolysis.
  • Panel A represents the xylose content of fibers from different segregating lines as indicated below the X-axis, after hydrolysis for 2 hrs in 2M TFA at 100°C. Also indicated is the copy number of the transgenic constructs.
  • WT wild type segregants - no transgenes.
  • Panel B represents the glucose content of hydrolysed fibers as in panel A. Note that line 7 is a transgenic line containing recombinant NodC and GFA transgenes as described in WOl 1/089021.
  • Panel C represents the xylose content of fibers from different lines and WT lines as indicated below the X-axis, after overnight hydrolysis in 1M TFA at 100°C.
  • FIG. 1 Comparison of the xylose level of sodiumhydroxide soluble versus insoluble fractions of cotton fibers from transgenic cotton comprising recombinant CslC4 and XT1 constructs after incubation in 1 M NaOH at 100°C for 1 hr.
  • Solid bar represents the fraction of xylose in the insoluble fraction, while the striped bar represents the fraction of xylose in the soluble fraction.
  • Different segregating transgenic cotton lines were tested.
  • HH homozygous transgenic plants.
  • NN segregants without the transgenes.
  • FIG. 1 Linkage analysis of hydrolyzed monosaccharides from cotton fibers obtained from a transgenic cotton line comprising recombinant CslC4 and XT1 constructs.
  • X axis various linkage forms of monosaccharides. Horizontally striped bars and dotted bars represent results from cotton fibers of null-segregants while the solid bars and diagonally striped bars represent results from cotton fibers of plants homozygous for the transgenes.
  • FIG. 4 Schematic representation of the dyeability of cotton fibers obtained from transgenic cotton lines comprising recombinant CslC4 and XTl constructs.
  • X-axis various segregants (null (N ) or homozygous for the transgenes (HH)) for different transgenic lines.
  • Y-axis % Exhaustion of the a direct dye (Sirius Red Violet RL) used for dying the cotton fibers.
  • Panel A dyeing conditions at pH 7.0;
  • Panel B dyeing conditions at pH 4.0.
  • FIG. 7 Comparison of the xylose level of sodiumhydroxide soluble versus insoluble fractions of mature cotton fibers from progeny lines (BC1 S2) of the transgenic cotton lines comprising recombinant CslC4 and XTl constructs after incubation in 1 M NaOH at 100°C for 1 hr.
  • the horizontally striped bar represents the fraction of xylose in the insoluble fraction, while the diagonally hatched bar represents the fraction of xylose in the soluble fraction.
  • Different progeny lines were tested.
  • HH homozygous transgenic plants.
  • NN plants without the transgenes.
  • Figure 8 Linkage analysis of hydrolyzed monosaccharides from cotton fibers obtained from BC1 S2 progeny of transgenic cotton lines comprising recombinant CslC4 and XTl constructs.
  • X axis various linkage forms of monosaccharides.
  • HH fibers from homozygous transgenic progeny
  • NN fibers from non-transgenic cotton plants.
  • Figure 9 Linkage analysis of hydrolyzed monosaccharides from cotton fibers obtained from BC1 S2 progeny of transgenic cotton lines comprising recombinant CslC4 and XTl constructs.
  • X axis various linkage forms of monosaccharides.
  • HH fibers from homozygous transgenic progeny
  • NN fibers from non-
  • Xylose (Panel A) and glucose (Panel B) levels (expressed as percentage) in developing fibers from transgenic cotton lines comprising recombinant CslC4 and XT1 constructs (HH) compared to developing fibers from non-trangenic control cotton plants (NN).
  • Figure 10 xXyll (Panel A) and 4xyll (Panel B) levels in developing fibers from transgenic cotton lines comprising recombinant CslC4 and XT1 constructs (HH) compared to developing fibers from non-trangenic control cotton plants (NN). Hatched bar; 10 dpa fiber; checkered bar: 15 dpa fiber; dotted bar: 20 dpa fiber; horizontally striped bar: 25 dpa fiber; solid bar: 30 dpa fiber.
  • FIG. 11 Schematic representation of the dyeability of cotton fibers obtained from transgenic cotton lines comprising recombinant CslC4 and XT1 constructs.
  • X-axis various transgenic lines (HH) or non-transgenic cotton lines (WT).
  • Y-axis % Exhaustion of the a reactive dye (Avitera®) used for dying the cotton fibers.
  • FIG. 12 Schematic representation of the dyeability of yarns from cotton fibers obtained from transgenic cotton lines comprising recombinant CslC4 and XT1 constructs.
  • X-axis various transgenic lines (HH) or non-transgenic cotton lines (NN).
  • Y- axis % Exhaustion of the a reactive dye (Avitera®) used for dying the yarns.
  • Checkered bars untreated yarn; hatched bars: scoured yarn; solid bars: mercerized yarn.
  • Figure 13 Color strength after treatment of yarns from cotton fibers obtained from transgenic cotton lines comprising recombinant CslC4 and XT 1 constructs.
  • X-axis various transgenic lines (solid bars) or non-transgenic cotton lines (hatched bars).
  • Plant fibers such as cotton fibers, obtainable from transgenic plants comprising a recombinant construct encoding cellulose synthase like C4 protein, preferably in combination with a recombinant construct encoding xyloglucan 6-xylosyltransferase, have altered fiber properties, including an increased dyeability i.e. an increase capacity for the uptake and retention of dyes, particularly for direct dyes. Without intending to limit the invention to a particular mode of action, it is thought that this increased dyeability might correlate with a decreased crystallinity and order structure of the cellulose network in the fibers, due to the increased presence of hemicellulose, such as xyloglucan. It is also expected that the fibers according to the invention have an increased capacity for the uptake of other molecules or chemicals.
  • the invention provides a method for providing plant fibers with altered dyeing properties of a plant fiber comprising the step of
  • ii a DNA region encoding a cellulose synthase-like C protein; and iii. optionally, a DNA region involved in transcription termination and polyadenylation; and
  • promoter denotes any DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription.
  • a promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymerase, and can comprise various other sites (e.g., enhancers), at which gene expression regulatory proteins may bind.
  • plant-expressible promoter means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S, the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters and the like.
  • the promoter may be a heterologous promoter not naturally associated with the DNA region operably linked to it, such as the DNA region coding for xyloglucan glycosyltransferases or xyloglucan 6- xylosyltransferases as described herein.
  • constitutive plant-expressible promoters may also be suitable for the invention.
  • constitutive promoters include the promoter from the actin gene (McElroy et al. (1990) Plant Cell 2: 163-171), the CaMV35S promoter (Odell et al. (1985) Nature 313: 810-812), the CaMV19S promoter (Nilsson et al. (1997) Physiol. Plant. 100: 456-462), the GOS2 promoter (de Pater et al. (1992) Plant. J. 2(6): 837-44), the promoter from ubiquitin gene (Christensen et al. (1992) Plant Mol. Biol.
  • inducible promoters such as a temperature inducible or a chemically inducible promoter or a promoter which is responsive to developmental cues, may be used in accordance with the invention. Tissue selective promoters may also be used.
  • a fiber-preferential or fiber-selective promoter is used.
  • the term "fiber specific” or “fiber cell specific” or “fiber-selective”, with respect to the expression of a gene or with respect to a promoter refers to, for practical purposes, the highly specific, expression of a gene or expression directed by a promoter, in fiber cells of plants, such as cotton plants. In other words, transcript levels of a DNA in tissues different of fiber cells is either below the detection limit or very low (less than about 0.2 picogram per microgram total RNA).
  • fiber-preferential or “fiber-cell preferential” with respect to the expression of a DNA in accordance with this invention, refers to an expression pattern whereby the DNA is expressed predominantly in fiber cells or fibers, but expression can be identified in other tissues of the plant.
  • the expression in fiber cells is about 2 to about 10 times higher in the fiber cells than in other tissues.
  • Such promoters include the promoter from cotton from a fiber-specific ⁇ - tubulin gene (as described in WO0210377), the promoter from cotton from a fiber-specific actin gene(as described in WO0210413), the promoter from a fiber specific lipid transfer protein gene from cotton (as described in US5792933), a promoter from an expansin gene from cotton (WO9830698) or a promoter from a chitinase gene in cotton (US2003106097) or the promoters of the fiber specific genes described in US6259003 or US6166294.
  • Fiber selective promoters as described in WO08/083969 (from cotton glucanase genes), WO 12/093032 (from cotton FS18 or SCW-PRP gene) or in US application 13/630,1 19 (from cotton FB8-like genes) are also suitable plant-expressible promoters.
  • the promoter comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide position 4208 to nucleotide position 5615 or having the nucleotide sequence of SEQ ID No. 5 from nucleotide position 75 to 1482.
  • a cellulose synthase-like C or "xyloglucan glycosyltransferase” is an enzyme which contributes to the synthesis of xyloglucan, particularly to the ⁇ -1,4 glucan backbone, transferring glucose residues from a nucleotide diphosphate sugar onto the glucan polysaccharide backbone.
  • Xyloglucan (XyG) is a major hemicellulose in the primary walls of many land plants. It has a P-l ,4-glucan backbone that is substituted in a regular pattern with xylosyl residues plus other sugars (including galactose and fucosyl) that vary depending on the plant species. In dicotyledons and non-graminaceous monocotyledons, xyloglucan is the major hemicellulosic polysaccharide and the principal polysaccharide that cross-links the cellulose microfibrils.
  • XyG is able to bind cellulose tightly because its -D-(l ,4)-glucan cellulose-like backbone can form numerous hydrogen bonds with the microfibrils, whereas the side chains give rise to regions where microfibril binding is interrupted.
  • XXXG the basic repeating xyloglucan subunit subunit is XXXG, which is composed of a P-l ,4-glucan, where three out of four glucosyl residues are linked to a-D- xylosyl residues at the 0-6 position.
  • Cellulose synthase-like C enzymes are Golgi membrane -bound proteins.
  • Cellulose synthase-like C proteins are characterized by being multi-transmembrane protein with a cytosolic active site.
  • E.g. CslC4 (SEQ ID No. 2) has transmembrane regions located at AA positions 90-110; 144-164; 469-489; 494-514; 623-643 an 648- 668.
  • the catalytic domain are predicted to have a D, D, D, Q/RXXRW motif common to the Cellulose Synthase-Like superfamily. Amino acid sequence from amino acid positions 119-431 of SEQ ID No.
  • Suitable proteins with cellulose synthase-like C (or xyloglucan glycosyltransferase) activity include CSL C4 from Arabidopsis thaliana (Accession Q9LJP4) or CLS C4 from Hordeum vulga -e (AV31214.1), TmCSL from nasturtium (Tropaeolum majus; SEQ ID NO. 6) or cellulose synthase like C4 from Physcomitrella patens (Accession EDQ51504) and the methods and means described herein comprise the use of nucleic acid regions encoding such polypeptides. Also encompassed is the use of a nucleic acid comprising the nucleotide sequence of SEQ ID No. 1 or the nucleotide sequence of SEQ ID No. 5 from nucleotide position 1485 to nucleotide position 3506.
  • Suitable polypeptides with cellulose synthase-like (or xyloglucan glycosyltransferase) actitivity comprising the following amino acid sequences can be found in various publicly available databases, including the following cellulose synthase- like C3 [Oryza sativa (japonica cultivar-group)] Accession: DAA01751.1 - GI: 34419220; cellulose synthase-like C2 [Oryza sativa (indica cultivar-group)] Accession: DAA01750.1 - GI: 34419218; cellulose synthase-like CI [Oryza sativa (japonica cultivar-group)] Accession: DAA01749.1 - GL34419216; cellulose synthase-like C3 [Physcomitrella patens] Accession: AB 155235.1 - GI: 1 14224789; cellulose synthase-like C2 [Physcomitrella pat
  • vesca Accession: XP_004291240.1 - GI: 470109929; xyloglucan glycosyltransferase 12-like [Fragaria vesca subsp. vesca] Accession: XP_004290998.1 - GI: 470109425; xyloglucan glycosyltransferase 6-like [Fragaria vesca subsp.
  • variants or homologues of the specifically mentioned xyloglucan glycosyltransferases which still have the enzymatic activity of transferring glucose residues from a nucleotide diphosphate sugar onto the -l ,4-glucan polysaccharide backbone.
  • variant xyloglucan glycosyltransferases proteins may contain insertions, substitutions or deletions of one or more amino acids in the specifically mentioned amino acid sequences of xyloglucan glycosyltransferases.
  • Amino acids of the polypeptide may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or ⁇ -sheet structures).
  • Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 1 of the present patent application).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions or deletions will usually be of the order of about 1 to 10 amino acid residues.
  • the variant proteins retain the conserved active site amino acid residues, and/or other conserved or semi-conserved regions, such as the ones for xyloglucan glycosyltransferases defined herein.
  • variants or homologues are orthologous sequences and paralogous sequences, which terms encompass evolutionary concepts to describe ancestral relationships of genes.
  • paralogous relates to gene-duplications within the genome of a species leading to paralogous genes. Paralogues of xyloglucan glycosyltransferases may easily be identified by performing a BLAST analysis against a set of sequences from the same species as the query sequence.
  • the term "orthologous” relates to homologous genes in different organisms.
  • Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include Ml 3 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site- directed mutagenesis protocols.
  • Encompassed by the invention are also essentially similar amino acid sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the xyloglucan glycosyltransferases amino acid sequences.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • the alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J. Mol Biol. 48: 443-453).
  • RNA sequences are to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • Suitable nucleotide sequences encoding a xyloglucan glycosyltransferase may comprise a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID No. 1 or with nucleotide sequence of SEQ ID No.
  • nucleotide position 1485 5 from nucleotide position 1485 to nucleotide position 3506, such as having at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the herein specifically mentioned xyloglucan glycosyltransferase encoding nucleic acids.
  • nucleotide sequences encoding a xyloglucan glycosyltransferase may comprise a nucleotide sequence hybridizing under stringent conditions with the herein specifically mentioned xyloglucan glycosyltransferase encoding nucleic acids.
  • hybridization is a process wherein substantially homologous complementary nucleotide sequences anneal to each other.
  • the hybridization process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • the hybridization process can also occur with one of the complementary nucleic acids immobilized to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • the hybridization process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilized by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • the stringency of hybridization is influenced by conditions such as temperature, salt concentration, ionic strength and hybridization buffer composition.
  • Stringent hybridization conditions means that hybridization will generally occur if there is at least 95%, for instance at least 97% sequence identity between the probe and the target sequence.
  • Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65 °C.
  • Other hybridization and wash conditions are well known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly chapter 1 1.
  • the recombinant DNA molecules as herein described optionally comprise a DNA region involved in transcription termination and polyadenylation.
  • a variety of DNA region involved in transcription termination and polyadenylation functional in plants are known in the art and those skilled in the art will be aware of terminator and polyadenylation sequences that may be suitable in performing the methods herein described.
  • the polyadenylation region may be derived from a natural gene, from a variety of other plant genes, from T-DNA genes or even from plant viral genomes.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or from any other eukaryotic gene.
  • the term "providing a recombinant DNA molecule” may refer to introduction of an exogenous DNA molecule to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell.
  • the term may also refer to introduction of the recombinant DNA molecule by crossing of a transgenic plant comprising the recombinant DNA molecule with another plant and selecting progeny plants which have inherited the recombinant DNA molecule or transgene.
  • Yet another alternative meaning of providing refers to introduction of the recombinant DNA molecule by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.
  • Transformation of plants is now a routine technique.
  • any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72- 74 ; Negrutiu et al. (1987) Plant. Mol. Biol.
  • Transgenic rice plants can be produced via Agrobacterium-mediated transformation using any of the well-known methods for rice transformation, such as described in any of the following: European patent application EP 1 198985 Al ; Aldemita and Hodges (1996) Planta 199: 612-617 ; Chan et al.
  • the recombinant DNA molecules according to the invention may be introduced into plants in a stable manner or in a transient manner using methods well known in the art.
  • the chimeric genes may be introduced into plants, or may be generated inside the plant cell as described e.g. in EP 1339859.
  • the methods for altering dyeing properties of a plant fiber through providing a first recombinant DNA molecule encoding a cellulose synthase-like C protein as herein described comprise the further step of providing a second recombinant DNA molecule, wherein the second recombinant DNA molecule comprising the following operably linked DNA molecules:
  • XXT xyloglucan 6-xylosyltransferase protein
  • Suitable xyloglucan 6-xylosyltransferases polypeptides comprising the following amino acid sequences can be found in various publicly available databases, including the following: xyloglucan 6-xylosyltransferase [Arabidopsis thaliana] Accession: AEE80384.1 - GL332646863; xyloglucan 6-xylosyltransferase [Arabidopsis thaliana] Accession: AEE80383.1 - GI: 332646862; xyloglucan 6-xylosyltransferase [Arabidopsis thaliana] Accession: NP 001030917.1 GI: 79316129; xyloglucan 6-xylosyltransferase [Arabidopsis thaliana] Accession: NP_567241.1 - GI: 1841 1962; xyloglucan 6- xylosyltransferase [Arabidopsis
  • xyloglucan 6-xylosyltransferase proteins comprise an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID No. 4 or with the amino acid sequence encoded by the nucleotide sequence of SEQ ID No. 5 from nucleotide position 5618 to nucleotide position 7000.
  • Encompassed by the invention are also essentially similar amino acid sequences having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the mentioned xyloglucan 6-xylosyltransferase amino acid sequences.
  • Suitable nucleotide sequences encoding a xyloglucan 6-xylosyltransferase may comprise a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID No. 4 or with nucleotide sequence of SEQ ID No.
  • nucleotide position 5618 5 from nucleotide position 5618 to nucleotide position 7000, such as having at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the herein specifically mentioned xyloglucan 6-xylosyltransferase encoding nucleic acids.
  • nucleotide sequences encoding a xyloglucan 6-xylosyltransferase may comprise a nucleotide sequence hybridizing under stringent conditions with the herein specifically mentioned xyloglucan 6-xylosyltransferase encoding nucleic acids.
  • the plant-expressible promoter of the second recombinant construct may be a constitutive promoter, an inducible promoter, a tissue specific promoter or a fiber-selective or fiber-preferential promoter as herein described elsewhere.
  • the invention thus provides a method to produce plants with increased xyloglucan level in the cell walls of cells of such plants comprising the steps of providing a first and second recombinant DNA molecule to at least one cell of a plant, wherein the first recombinant DNA molecule comprises the following operably linked DNA molecules:
  • ii a DNA region encoding a cellulose synthase-like C protein; and iii. optionally, a DNA region involved in transcription termination and polyadenylation;
  • the second recombinant DNA molecule comprises the following operably linked DNA molecules:
  • XXT xyloglucan 6-xylosyltransferase protein
  • Plant cell walls such as plant cell walls in fibers, including cotton fibers obtained from the above mentioned plants are characterized in having a xylose level (after hydrolysis of the polysaccharides, such as hydrolysis by treatment with trifiuoro-acetic acid (TFA) for at least 2 hours) which is may be more than 10 times higher than plant cells walls, including in fibers, from plants without the herein described transgenes, while glucose levels remain similar (particularly for plant cell walls with a high proportion of secondary cell walls)
  • xylose levels are higher than 1% of fiber weight but may be higher than 1 , 5%, 2% or 2.5% after 2hr hydrolysis in TFA.
  • the obtained xylose is further characterized by being located in alkaline-insoluble fraction (aOH insoluble fraction) which is indicative of the xylose being located in the secondary cell wall.
  • the increased xylose can further be characterized as being terminal xylose (xXyll) (as opposed to 4Xyll or 3,4Xyll typically resulting from xylan hydrolysis).
  • Another embodiment of the invention are therefore plant cell walls, including fibers such as cotton fibers comprising an increased level of xyloglucan compared to fibers from unmodified plant not comprising a first, or a first and second recombinant construct as herein described.
  • the invention also provides plant cell walls, including fibers such as cotton fibers comprising a xylose level, preferably terminal xylose, of more than 1% after 2 hours of hydrolysis by trifluoroacetic acid.
  • Fibers as herein described, including cotton fibers, as produced by the methods according to the invention or obtained from the transgenic plants as described herein are also encompassed by the invention.
  • Such fibers are further characterized by their increased dyeability, i.e. their capacity to absorb and retain dye, including direct dyes, but it is also expected and demonstrated that the fibers have an increased capacity to absorb and retain fiber-reactive dyes. This capacity can be measured by measuring the exhaustion of dye from the dyebath.
  • direct dyes are dyes that are applied directly to the substrate in a neutral or alkaline bath. They produce full shades on cotton and linen without mordanting and can also be applied to rayon, silk, and wool. Direct dyes give bright shades but exhibit less wash-fastness. Various after-treatments are used to improve the wash- fastness of direct dyes, and such dyes are also referred to as "after-treated direct colors.” Direct Dyes adhere to the fabric molecules without help from other chemicals. Direct dyes are defined as anionic dyes with substantivity for cellulosic fibres, normally applied from an aqueous dyebath containing an electrolyte, usually sodium chloride ( aCl) or sodium sulfate ( a2S04).
  • Chemically direct dyes are salts of complex sulfonic acids, mostly unmetallised azostructures, but they may also contain di-azo or poly-azo structures. Numerous direct dyes and their characteristics can be found on www.worddyevariety .com. One of the direct dyes used herein as example is Sirius Red Violet.
  • Fiber-reactive dyes are molecules that combine chromophores with a reactive group that forms strong covalent bonds with the fiber via reaction with hydroxyl groups.
  • the covalent bonds provide a good resistance of the dyed fiber against laundring. Colorfastness can be improved using cationic fixatives.
  • the dyes contain a reactive group (often trichlorotriazine), usually a haloheterocycle or an activated double bond, that, when applied to a fibre in an alkaline dye bath, forms a chemical bond with an hydroxyl group on the cellulosic fiber.
  • Reactive dyes are categorized by their functional group and may contain as functional group a haloheterocycle including monochlorotriazine, Monofiuorochlorotriazin, dichlorotriazine, difluorochloropyrimidine, dichloroquinoxaline or trichloropyrimidine, or a activated double bound including vinylsulfone and vinylamine.
  • Fiber-reactive dyes may also be bifunctional, i.e. comprising more than one, different reactive groups.
  • Fibers or yarns made of fibers according to the invention may be used to improve the wash out of colors on other items in the same laundry. They may also be used to improve fading resistance.
  • the fibers according to the invention have a higher capacity to absorb or adsorb chemical substances other than dyes, including e.g. drugs.
  • the fibers and yarns and fabrics according to the invention may thus be used
  • Fibers according to the invention, or material containing such fibers may also be used to absorb contaminants/infectious organisms from e.g. wounds and send of patch for laboratory for analysis e.g. for patient follow-up. Fibers according to the invention, or material containing such fibers may also be used to absorb moisture and liquids in packaged food products, such as packaged meat products.
  • Cotton fibers obtained from transgenic cotton plants as described herein are also characterized by having often a lower micronaire than cotton fibers obtained from isogenic plants without the transgenes.
  • Micronaire is an indicator of air permeability. It is regarded as an indicator of both fineness (linear density) and maturity (degree of cell-wall development). Fineness is generally expressed as gravimetric fineness or linear density (wall area x a constant) and maturity is generally expressed as maturity ratio (wall area divided by perimeter square).
  • ASTM D1448 describes a standard test method for Micronaire reading of cotton fibers.
  • a "fiber” is botanically defined as a long narrow tapering cell, dead and hollow at maturity with a rigid thick cell wall composed mostly of cellulose and usually lignin.
  • Soft or bast fibers are found in the phloem (inner bark) of dicotyledonous stems (flax, jute, hemp, ramie). Hard or leaf fibers are found in monocot leaf vascular bundles (sisal, manilla hemp, pineapple). Surface fibers grown from the surface of seeds (cotton), leaves or fruits (coconut coir).
  • Cotton fiber refers to a seed trichome, more specifically a single cell of a fiber-producing plant, such as cotton, that initiates from the epidermis of the outer integument of the ovules, at or just prior to anthesis.
  • a fiber-producing plant such as cotton
  • Cotton fibers in particular from Gossypium hirsutum, undergo four overlapping developmental stages: fiber cell initiation, elongation, secondary cell wall biosynthesis, and maturation.
  • Fiber cell initiation is a rapid process.
  • White fuzzy fibers begin to develop immediately after anthesis and continue up to about 3 days post-anthesis (DPA), which is followed by fiber cell elongation (until about 10 to about 17 DPA).
  • DPA days post-anthesis
  • secondary cell wall biosynthesis initiates and continues to about 25 to about 45 DPA, followed by a maturation process from about 45 to about 60 DPA.
  • the secondary cell wall synthesis and maturation phase are herein commonly referred to as "fiber strength building phase".
  • fiber strength building phase Only about 25 to 30% of the epidermal cells differentiate into the commercially important lint fibers (Kim and Triplett, 2001). The majority of cells does not differentiate into fibers or develop into short fibers or fuzz.
  • the fiber cells elongate rapidly, synthesize secondary wall components, and show dramatic cellular, molecular and physiological changes. Fiber elongation is coupled with rapid cell growth and expansion (Seagull, 1991. In Biosynthesis and biodegradation of cellulose (Haigler, C. H. & Weimer, P. J., eds) pp.
  • a "fiber-producing plant” refers to a plant species that produces fibers, such as a cotton plant.
  • Gossypium species the A genome diploid Gossypium species and AD genome allotetraploid Gossypium species are known to produce spinnable fiber.
  • AADD Gossypium hirsutum
  • This group is known in the United States as American Upland cotton, and their fibers vary in length from about 7/8 to about 1 5/16 inches (about 22 - about 33 mm).
  • a third group, G. herbaceum (AA) and G. arboreum (AA) embraces cotton plants with fibers of shorter length, about 1/2 to about 1 inch (about 13 - about 25 mm), that are native to India and Eastern Asia. None from this group is cultivated in the United States. [89] The invention also relates to yarns made from the fibers according to the invention, as well as fabrics made from those yarns.
  • recombinant genes are provided as herein described, in particular where such recombinant genes comprise a heterologous plant-expressible promoter, particularly where the heterologous promoter is a fiber- selective or fiber-preferential promoter.
  • the term "gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an R A molecule (e.g. into a pre-mRNA, comprising intron sequences, which is then spliced into a mature mR A, or directly into a mR A without intron sequences) in a cell, operable linked to regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (with introns) and a 3 ' non-translated sequence comprising e.g. transcription termination sites.
  • cDNA sequence refers to a nucleic acid sequence comprising the 5' untranslated region, the coding region without introns and the 3 ' untranslated region and a polyA tail.
  • Endogenous gene is used to differentiate from a “foreign gene”, “transgene” or “chimeric gene”, and refers to a gene from a plant of a certain plant genus, species or variety, which has not been introduced into that plant by transformation (i.e.
  • transgene it is not a "transgene", but which is normally present in plants of that genus, species or variety, or which is introduced in that plant from plants of another plant genus, species or variety, in which it is normally present, by normal breeding techniques or by somatic hybridization, e.g., by protoplast fusion.
  • an "endogenous allele" of a gene is not introduced into a plant or plant tissue by plant transformation, but is, for example, generated by plant mutagenesis and/or selection, introgressed from another plant species by, e.g., marker-assisted selection, or obtained by screening natural populations of plants.
  • “Expression of a gene” or “gene expression” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA molecule. The RNA molecule is then processed further (by post-transcriptional processes) within the cell, e.g. by RNA splicing and translation initiation and translation into an amino acid chain (polypeptide), and translation termination by translation stop codons.
  • the term “functionally expressed” is used herein to indicate that a functional, i.e. biologically active, protein is produced; the term “not functionally expressed” to indicate that a protein with significantly reduced or no functionality (biological activity) is produced or that no or a significantly reduced amount of protein is produced.
  • the invention is also directed towards fiber-producing plants comprising one or more recombinant construct according to the invention.
  • Preferred fiber-producing plants include cotton.
  • Cotton as used herein includes Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum.
  • Cotton progenitor plants include Gossypium arboreum, Gossypium herbaceum, Gossypium raimondii, Gossypium longicalyx and Gossypium kirkii.
  • the methods and means of the current invention may also be employed for other plant species such as hemp, jute, flax and woody plants, including but not limited to Pinus spp., Populus spp., Picea spp., Eucalyptus spp. etc.
  • the plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert and are also encompassed by the invention.
  • plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • the plant cell according to the invention is non-propagating or cannot be regenerated into a plant.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
  • SEQ ID No. 1 nucleotide sequence of xyloglucan glycosyltransferase 4 from
  • SEQ ID No. 2 amino acid sequence of xyloglucan glycosyltransferase 4 from
  • SEQ ID No. 3 nucleotide sequence of xyloglucan 6-xylosyltransferase from Arabidopsis thaliana.
  • SEQ ID No. 4 amino acid sequence of xyloglucan 6-xylosyltransferase from Arabidopsis thaliana.
  • SEQ ID No. 5 nucleotide sequence of T-DNA vector comprising recombinant CSLc4 and XXTl constructs.
  • SEQ ID No. 6 amino acid sequence of xyloglucan glycosyltransferase from Tropaeolum majus.
  • Example 1 Generation of transgenic cotton plants expressing AtCSLC4 and AtXTl.
  • a recombinant xyloglucan glycosyltransferase comprising the following DNA regions:
  • a recombinant xyloglucan 6-xylosyltransferase comprising the following DNA regions:
  • the nucleotide sequence of the T-DNA vector is represented in SEQ ID No. 5.
  • the nucleotide positions of the different elements are represented in Table 2.
  • This T-DNA vector was introduced into Agrobacterium tumefaciens strains containing a helper Ti-plasmid and used in cotton transformation essentially as described in WO00/71733. Several transgenic lines comprising the recombinant genes in one or more copies were identified. The regenerated cotton plants were selfed or crossed with non-transformed Coker plants, and fibers from the progeny plants were analyzed as described in the following examples. Total R A was extracted from developing fibers (between 25 and 30 dpa) from 10 TO plants and analyzed by Northern blot for transgene expression.
  • Increased 6,4Glcl was not consistently linked to the presence of the transgenes as a significant part of this fraction may be derived from partial derivisation of cellulose in addition to the fraction derived from xyloglucan.
  • the increase xylose level in cotton fibers from NodC and GFA expressing plants was primarily due to 4Xyll, indicating increased xylan levels instead of xyloglucan.
  • Figure 10 shows the results of the additional linkage analysis of the saccharides in the developing fibers, demonstrating that the increased xylose level in developing fibers is primarily terminal xylose.
  • the terminal xylose (xXyll) is increased more than 20-fold in the fibers from transgenic cotton plants compared to fibers from non-transgenic cotton plants, whereas 4Xyll level is increased only two to three-fold. No significant increase could be observed in xGal and xFucl .
  • Example 3 Increased dyeabili y of fibers from transgenic cotton plants expressing AtCSLC4 and AtXTl.
  • Fibers from transgenic lines expressing AtCSLC4 and AtXTl exhibited a 30 to 75% higher exhaustion than fibers from null segregants.
  • the uptake of direct dyes is representative for the capacity of the engineered cotton fibers to take up other chemical substances, including chemical substances used to modify the end-use properties of cotton fabric made from such fibers, such as flame - retardants, wrinkle-free agents, softeners. These fibers may thus be more easily modified with these finishing agents as well.
  • Cotton fibers from transgenic plants expressing CslC4+XTl transgenes, as well as cotton fibers from non-transgenic cotton plants, and yarns of such fibers (either untreated or scoured or mercerized) were also tested for uptake of reactive dyes such as Avitera® Red ( more info is available e.g. on webpage www.swisscolor.sk/sites/default/files/sites/default/files/pdf/avitera_red_se.pdf).
  • the yarns were either untreated, scoured or mercerized.
  • the yarns were treated in a solution of 5g/L sodium carbonate, 07g/L crossCOLTOR PTM using a yarn/water ratio of lg/ 20 mL solution.
  • the yarns were treated at 95°C for 30 minutes and then rinsed with demineralized water for 10 min at 60°C, followed by cold rinsing with demineralized water to which 1 ml/L acetic acid was added and a final rinse with demineralized water.
  • the fibers were treated in a solution of 20°Be NaOH using a yarn/water ratio of lg/50 mL solution.
  • the solution was kept at 25°C for 30 min and rinsed with demineralized water once, followed by cold rinsing with demineralized water to which 5 ml/L acetic acid was added, and a final rinse with demineralized water.

Abstract

La présente invention concerne des méthodes d'augmentation du niveau des taux de xyloglucanes dans les parois de cellules végétales, y compris des parois de cellules végétales secondaires telles que celles observées dans des fibres naturelles ou des plantes produisant des fibres par expression d'une xyloglucane glycotransférase ou par expression combinée de xyloglucane glycotransférase et de xyloglucane xylotransférase. Les taux plus élevés de xyloglucanes mènent à une capacité accrue d'absorption et de rétention de colorants.
PCT/EP2014/062363 2013-06-14 2014-06-13 Fibres végétales à propriétés de coloration améliorées WO2014198885A1 (fr)

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