WO2016131951A1 - Plantes de solanum tuberosum pour la production de biocarburant - Google Patents

Plantes de solanum tuberosum pour la production de biocarburant Download PDF

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WO2016131951A1
WO2016131951A1 PCT/EP2016/053541 EP2016053541W WO2016131951A1 WO 2016131951 A1 WO2016131951 A1 WO 2016131951A1 EP 2016053541 W EP2016053541 W EP 2016053541W WO 2016131951 A1 WO2016131951 A1 WO 2016131951A1
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plant
gene
nucleic acid
enzyme
lignin
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PCT/EP2016/053541
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English (en)
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Stephan POLLMANN
Jesús Vicente CARBAJOSA
Joaquín MEDINA
Julia KEHR
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Repsol, S.A.
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Publication of WO2016131951A1 publication Critical patent/WO2016131951A1/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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)

Definitions

  • the present invention relates to the field of agriculture and of energy (biofuel) production.
  • biofuel biofuel
  • plants that have been modified to be cropped and used as source of fermentable sugars are produced by plants that have been modified to be cropped and used as source of fermentable sugars.
  • Bioethanol is an alcohol made by fermentation, mostly from carbohydrates produced in sugar or starch crops such as corn, sugarcane, or sweet sorghum.
  • Cellulosic biomass derived from non-food sources, such as trees and grasses, is also being developed as a feedstock for ethanol production.
  • bioethanol is mainly produced from lignocellulose biomass from urban waste and from agricultural residuals. Ethanol can be used as a fuel for vehicles in its pure form or as additive in fuel. Bioethanol is widely used in the USA and in Brazil.
  • the main sugar source from plants is the cell wall.
  • the contents of sugars in the cell wall are approximately the 75 % by weight.
  • Usually of the extractable sugars are disposed in chains of polysaccharides such as cellulose.
  • Cellulose is the polymer of ⁇ (1 ⁇ 4) linked D-glucose.
  • cellulose, hemicellulose, pectin and lignin form also part of the cell wall.
  • lignin that forms a rigid polymeric structure acts as cell cement (binder) and hampers glucose extraction from vegetal biomass.
  • Lignin is present in any plant type. It is a high molecular weight polymer made of monolignols (also named
  • phenylpropanoids including coniferyl alcohol (G), sinapyl alcohol (S) and paracoumaryl alcohol (H). These monolignols are synthesized inside the cell
  • FIG. 1 shows the lignin biosynthesis pathway with the enzymes and reactions involved. Biosynthesis of monolignols implies a battery of enzymes in order to transform phenylalanine (Phe) to the three different monolignols.
  • plants such as food crops, trees and grasses have been genetically modified (by means of sited mutagenesis or by transformation) in order to alter their lignocellulosic composition (i.e.. composition of cellulose, hemicellulose and lignin of the plant cell wall), and so facilitating sugar extraction and fermentation.
  • lignocellulosic composition i.e.. composition of cellulose, hemicellulose and lignin of the plant cell wall
  • modified plants to increase sugar extraction include the patent application document WO2008064289 (The Samuel Roberts Nobel
  • alfalfa is genetically modified by RNAi silencing of genes coding for enzymes of the lignin biosynthesis pathway, namely C4H, HCT3a, C3N, CCoAOMT, F5H and caffeic acid/5-hydroxyferulic acid O- methyltransferase (COMT).
  • Sugar extraction percentages are shown in relation with the control (wild-type) alfalfa and an evident increase in the percentage of extraction can be observed when C4H and CCoAOMT are silenced.
  • particular ratios of S/G are indicated.
  • Down-regulation of COMT and CCoAOMT are proposed since they provide direct gains in saccharification efficiency in the absence of biomass yield reduction.
  • RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass
  • Plant Biotechnology Journal - 2012, Vol. No. 10, pp.: 1067-1076 shows in sugarcane a way of silencing COMT by means of RNAi for reducing lignin content and improving so the yield of directly fermentable glucose from lignocellulosic biomass up to 29 % and 34 %.
  • This waste material is the one to be used for sugar extraction.
  • Fornale et al. disclose the differential impact on phenotype caused in different organs by the silencing (down-regulation by means of RNAi) of CAD enzyme coding gene in maize (Zea Maize; corn). In any case, sugar bioavailability is increased in transgenic (transformed) plants while lignin content is maintained or reduced, depending on the assayed organ, without compromising the plant or without affecting growth performance. Cellulosic bioethanol production from transgenic plants was 40 % to 51 % higher than production in wild-type plants. Fornale et al.
  • transgenic and wild-type plants are apparently identic and propose the repression level of CAD activity as a main cause for explaining the different observed effects in the lignin contents as well as in its final monolignol composition.
  • Fornale et al. are silent about any effect of CAD silencing in cob, which is the maize part mainly used for food.
  • Inventors developed genetically modified plants of the Solanum tuberosum specie having a lignin composition of the cell wall with a particular range of the ratio between the monolignol monomers sinapyl alcohol (S) and coniferyl alcohol (G). This ratio is commonly known as S/G ratio.
  • S/G ratios of the plants of the invention lead to plants in which the cell wall preserves its functionality, while allowing extraction of glucose and of other fermentable sugars per mass of cell wall with high yields.
  • glucose extraction also named glucose extractability
  • glucose extraction is increased at least from 68 % to 846 % (thus approximately from 60 % to 900 %) in relation to the glucose extraction in the non-genetically modified plant (wild-type).
  • the invention relates to genetically modified plants of
  • Solanum tuberosum comprising a lignin composition with a ratio of the amounts of the monolignols sinapyl alcohol (S) / coniferyl alcohol (G) in the cell wall from 0.10 to 0.50, each monolignol amount being expressed as picomols of monolignol/milligram of cell wall.
  • a third aspect of the invention is a genetically modified plant cell of a plant or part of the plant of Solanum tuberosum as defined above, the cell comprising a nucleic acid gene silencing construct capable of down-regulating the expression of a gene coding for an enzyme of the lignin biosynthesis pathway.
  • Plant calli may be induced from these transgenic cells with appropriate culturing mediums, as well as explants with other appropriate culturing mediums.
  • the transgenic cells may also be reproductive cells also named gametophytes derived from stamens and styles of the plant.
  • calli masses of unorganized parenchyma cells derived from plant tissue (explants)
  • explants produced from the transgenic plant cell according to the third aspect of the invention.
  • Yet another aspect of the invention is a manufactured plant product of the plant or part of the plant of Solanum tuberosum as defined above, said manufactured plant product selected from the group consisting of milled parts of the plant, ground parts of the plant, and packaged parts of the entire aerial part of the plant.
  • Any of these manufactured plant products may then be processed for extracting glucose and other fermentable sugars, and then proceed with any suitable fermentation process for obtaining bioethanol.
  • Another aspect (fifth) of the invention is a method of producing the plant as defined above, comprising:
  • tuberosum plant with a nucleic acid gene-silencing construct capable of down- regulating the expression of a gene coding for an enzyme of the lignin biosynthesis pathway, to obtain a transformed cell and or tissue;
  • Another aspect (sixth aspect) of the invention is a method of producing bioethanol comprising:
  • the invention relates also to a particular interference nucleic acid gene- silencing construct that down-regulates the expression of cinnamoyl CoA reductase and/or cinnamyl alcohol dehydrogenase, the construct comprising nucleotide sequences selected from SEQ ID NO: 1 and SEQ ID NO: 3.
  • FIG. 1 shows schematically the lignin biosynthesis pathway, leading to H- lignin, S-lignin or G-lignin, depending on the abundance of the monolignol monomers in the final lignin polymer.
  • FIG. 2 Schematic representation of the cassette (nucleic acid gene silencing construct) integrated in potato transgenic plants.
  • FIG. 2A the construct for silencing expression of the CAD2 enzyme is represented.
  • the construct for silencing expression of the CCR1 enzyme is shown in FIG. 2B.
  • Each figure shows the nucleic acid gene silencing construct in a binary vector (called pSP- DEST1 -CAD2 in FIG. 2A; and pSPDEST1 -CCR1 in FIG. 2B.
  • the construct in the vector has been amplified between dashed lines.
  • attB1 and 2 are recognition sites in the GATEWAYTM cloning system.
  • Kan (r) refers to the kanamycin resistance mediating NPTII sequence.
  • T-DNA RB and T-DNA LB are T-DNA left and right border repeat sequences defining and delimiting T-DNA border repeat sequences of Ti- and Ri-plasmids.
  • NOS_P and NOS_T depict nopal ine synthase promoter and terminator, respectively. Bar means the gene conferring resistance to the herbicide bialaphos (bar) that was originally cloned from Streptomyces hygroscopicus.
  • pA g7 is a fragment deriving from the binary pBIG plasmid (Becker et al. 1992, see below).
  • CAMV 35S is the constitutive cauliflower mosaic virus 35S promoter.
  • PDK and Cat are intron sequences from from pyruvate dehydrogenase kinase and catalase, respectively.
  • Terminator is a termination sequence.
  • SK is a fragment of pBlueScript SK(+) (Stratagene).
  • FIG. 3 is a bar diagram with the silencing levels of target genes CAD2 (panel A) and CCR1 (panel B) in several RNAi lines. Numbers of the lines are indicated at the top of the bars. Circled lines were those lines further analysed for metabolomic pattern. Y-axis shows the relative expression (2 _ ⁇ method).
  • FIG. 4 shows pictures of genetically modified potato (Solanum tuberosum) plants grown in the greenhouse.
  • CAD2 and CCR1 -RNAi lines are examples of plants showing normal phenotypes compared to wild-type Desiree plants.
  • WT means wild-type (non-transgenic plant for any of the assayed enzymes).
  • CAD2-iRNA lines and CCR1 -iRNA lines indicate, respectively, the lines down- regulated for CAD2 gene and for CCR1 gene.
  • FIG. 5 is another picture for showing the aspect of collected tubers from different RNAi genetically modified lines (CAD2 and CCR1).
  • FIG. 6 shows bar diagrams with the lignin content (FIG. 6 A) in g of lignin per mg of cell wall, glucose extractability (FIG. 6 B) in pmols of glucose per mg of cell wall, and the monolignol ratios (FIG. 6 C) indicated as relative abundance of each monolignol, in genetically modified plants in which G4D2 gene has been down-regulated.
  • X-axis (G) is the particular assayed genotype or lines. WT means wild-type.
  • FIG. 7 includes also a bar diagrams with the lignin content (FIG. 7 A) in g of lignin per mg of cell wall, glucose extractability (FIG. 7 B) in pmols of glucose per mg of cell wall, and the monolignol ratios (FIG. 7 C) indicated as relative abundance of each monolignol, in genetically modified plants in which CCR1 gene has been down-regulated.
  • X-axis (G) is the particular assayed genotype or lines. WT means wild-type.
  • Gene expression is to be understood as the process by which information from a gene is used in the synthesis of a functional gene product.
  • These gene products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA
  • RNA Ribonucleic acid
  • snRNA RNA splicing gene
  • translation RNA splicing
  • post-translational modification of a protein.
  • gene expression is the most fundamental level at which the genotype gives rise to the phenotype.
  • the genetic code stored in DNA is "interpreted" by gene expression, and the properties of the expression give rise to the organism's phenotype.
  • Such phenotypes are often expressed by the synthesis of proteins that control the organism's shape, or that act as enzymes catalysing specific metabolic pathways characterising the organism.
  • a “genetically modified plant”, also termed a “transgenic plant” (both
  • GMO genetically modified organism
  • DNA, RNA biotechnology new exogenous nucleic acid
  • This new exogenous nucleic acid performs in the host cell the desired function, being either the "knock-down" of endogenous genes; the silencing (or loss of expression or down-regulation) of endogenous genes, for example by interfering with the translation of this endogenous one; or the removal of exons to obtain a nonfunctional protein, among other possible functions.
  • New nucleic acid in particular DNA
  • DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence.
  • it can be synthesized, and then inserted into the host organism.
  • nucleic acid gene silencing construct a nucleic acid (DNA, RNA) construct that interferes with the expression of one or more genes by down-regulating or entirely
  • Down-regulation of a gene relates to the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable.
  • down-regulation is a process resulting in decreased gene and corresponding protein expression.
  • RNA silencing may be accomplished with constructs including or delivering in a cell double-stranded RNA (dsRNA), a DNA directed RNA interference (ddRNA), and small interfering RNA (siRNA).
  • dsRNA cell double-stranded RNA
  • ddRNA DNA directed RNA interference
  • siRNA small interfering RNA
  • nucleic acid gene silencing constructs induce one way or another degradation of messenger RNA.
  • the nucleic acid gene silencing constructs may also comprise promoters operatively linked to the gene sequences or fragments thereof coding for the enzymes that wished to be interfered (or silenced). Operatively linked” means that the gene sequence is disposed near the promoter sequence or relatively near in case additional restriction sites, termination sites or other elements stabilizing the construct are present.
  • the nucleic acid gene silencing constructs may also comprise resistance sequences (i.e. to predetermined antibiotics) that the skilled man in the art will know.
  • Particular promoters usually used for the expression of plant genes in constructs include the constitutive cauliflower mosaic virus 35S promoter (CaMV 35S).
  • constructs may form part of vectors or which is the same, be comprised in vectors.
  • vectors include not only bacterial plasmids, but also bacteria such as Escherichia coli and Agrobacterium tumefaciens.
  • transformation is the genetic alteration of a cell resulting from the direct uptake
  • exogenous genetic material exogenous DNA
  • Transformation is also used to describe the insertion of new genetic material into plant cells; Introduction of foreign DNA into eukaryotic cells is often called "transfection”.
  • stringent conditions mean conditions under which only nucleic acid base sequences coding for a polypeptide with any of CAD2 or CCR1 activity equivalent to the CAD2 or CCR1 enzyme genes encoded by a specific CAD2 or CCR1 enzyme gene sequence, can form hybrids with the specific sequence (referred to as specific hybrids), meanwhile nucleic acid base sequences coding for polypeptides with no such equivalent activity, do not form hybrids with the specific sequence (referred to as non-specific hybrids).
  • specific hybrids nucleic acid base sequences coding for polypeptides with no such equivalent activity
  • non-specific hybrids referred to as non-specific hybrids.
  • the invention relates to genetically modified plants of the Solanum tuberosum comprising a lignin composition of the aerial part of the plant (defined generally by stems and leaves) with a ratio of the amounts of the monolignols sinapyl alcohol (S) / coniferyl alcohol (G) in the cell wall from 0.10 to 0.50, each monolignol amount being expressed as picomols of monolignol/nnilligrann of cell wall.
  • the aerial part of the plant is the internodes of stem, and more particularly the third internode.
  • the Solanum tuberosum plant comprises plant cells, said cells comprising a nucleic acid gene construct capable of down-regulating the expression of a gene coding for an enzyme of the lignin biosynthesis pathway.
  • the Solanum tuberosum plant is genetically modified by down-regulation of the expression of a gene coding for an enzyme of the lignin biosynthesis pathway by means of biotechnology-inserted new exogenous nucleic acid (DNA, RNA) inserted in the host genome or in the cytoplasm of the cells (in this invention, plant cells).
  • the nucleic acid gene silencing construct capable of down-regulating the gene coding for the enzyme comprises a nucleotide sequence of a gene of the Solanum tuberosum coding for an enzyme of the lignin pathway synthesis; or comprises a nucleotide sequence with at least 80 % of identity with said nucleotide sequence of a gene of the Solanum tuberosum coding for an enzyme of the lignin pathway synthesis.
  • Solanum tuberosum plants are herbaceous perennials that grow about 60 cm (24 inch) high, depending on variety, with the culms dying back after flowering, fruiting and tuber formation. After flowering, potato plants produce small green fruits that resemble green cherry tomatoes, each containing about 300 seeds. Like all parts of the plant except the tubers, the fruit contain the toxic alkaloid solanine and are therefore unsuitable for consumption. All new potato varieties are grown from seeds, also called “true potato seed TPS or "botanical seed” to distinguish it from seed tubers. Plants propagated from tubers are clones of the parent, whereas those propagated from seed produce a range of different varieties.
  • the Solanum tuberosum plant is a genetically modified Solanum tuberosum derived from genetically modification of a plant of any variety consisting of any available elite germline that can be genetically manipulated in the same way disclosed in the invention.
  • the genetically modified plant derives from the Desiree plant variety.
  • the plant comprises an S/G ratio in the cell wall from 0.20 to 0.40, each monolignol amount expressed as picomols of monolignol (pmols)/ milligram (mg) of cell wall.
  • the S/G ratio in the cell wall is from 0.25 to 0.35.
  • resulting plant of the Solanum tuberosum comprises an S/G ratio that is decreased from 1 .0 to 7.0 times in relation to a wild type plant of the Solanum tuberosum, in which the nucleic acid gene silencing construct is absent.
  • the nucleic acid gene silencing construct is selected from the group consisting of an antisense-DNA, an interference RNA coding-DNA (RNAi), and combinations thereof.
  • the nucleic acid gene silencing construct is an interference RNA coding-DNA.
  • This interference RNA coding-DNA gives rise to an interference RNA selected from the group consisting of dsRNA, ddRNA and siRNA.
  • the enzyme is cinnamoyl CoA reductase and the nucleic acid gene silencing construct comprises a sequence selected from the group consisting of:
  • Preferred identity ranges with the nucleotide sequence comprising SEQ ID NO: 1 include 80-99 %, 90-99 %, 95-99%, preferably 95%, 96 %, 97 %, 98 %, or 99 %.
  • the identity between two amino acid sequences is preferably determined by using the BLASTP algorithm disclosed in Altschul, S. F., et. al. "Gapped BLAST and PSI-BLAST: a new generation of proteina database search programms", Nucleic Acids Research - 1997, Vol. No. 25, pp.: 3389 -3402, and NCBI http://www.ncbi.nlm.nih.gov/BLAST.
  • the nucleotide sequence of (a) consists in SEQ ID NO: 1 .
  • the nucleic acid gene silencing construct comprises among other sequences, SEQ ID NO: 1 .
  • SEQ ID NO: 1 corresponds to the entry PGSC0003DMP400034431 from database Phytozome (version 9.1 , at filing date retrievable from
  • CCR1 isoform 1 of Cinnamyl-CoA-reductase enzyme.
  • Cinnamoyl-CoA- reductase EC 1 .2.1 .44 catalyses the reaction cinnamaldehyde + CoA + NADP + v-cinnamyl-CoA + NADPH + H + .
  • CCR1 is isogene 1 of two potential CCR genes identified taken.
  • SEQ ID NO: 2 corresponds to the translated amino acid sequence of the enzyme.
  • the nucleic acid gene silencing construct comprises at least twice any of the sequences as defined in (a) or (b) above, wherein these at least two sequences are, in another particular embodiment disposed in sense and antisense orientations, and they are optionally separated by intron fragments.
  • the nucleic acid gene silencing construct is an interference RNA coding-DNA that comprises two copies of SEQ ID NO: 1 separated by an intron fragment, and gives raise when transcribed to a dsRNA.
  • This dsRNA incorporates into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the nucleic acid gene silencing construct comprises SEQ ID NO: 16.
  • SEQ ID NO: 16 is a sequence of 4081 nucleotides comprising two copies of SEQ ID NO: 1 in sense (from nucleotide 1451 to nucleotide 1801 of SEQ ID NO: 16) and antisense orientation (from nucleotide 2920 to nucleotide 3270 of SEQ ID NO: 16).
  • SEQ ID NO: 16 comprises a nucleotide sequence which is an intron fragment, that comprises a PDK intron (from pyruvate dehydrogenase kinase) and a Cat intron (from catalase).
  • This intron fragment goes from nucleotide 1886 to nucleotide 2863 of SEQ ID NO: 16. From nucleotide 31 to nucleotide 1376 of SEQ ID NO: 16 there is the sequence of the cauliflower mosaic virus promoter 35S (CaMV35S). A terminator sequence (NOS_T from nopaline synthase) is from nucleotide 3287 to nucleotide 4051 of SEQ ID NO: 16. Finally attB sequences are present at 5'- and 3'-ends, namely an attB1 from nucleotide 5 to 29 of SEQ ID NO: 16; and an attB2 (in anti-sense orientation) from nucleotide 4053 to nucleotide 4077 of SEQ ID NO: 16. These attB sequences allow the cloning in a
  • the enzyme is cinnamyl alcohol dehydrogenase and the nucleic acid gene silencing construct comprises a sequence selected from the group consisting of:
  • nucleic acid gene silencing construct comprises, among other sequences, SEQ ID NO: 3.
  • SEQ ID NO: 3 corresponds to the entry PGSC0003DMP400044639 from database Phytozome (version 9.1 , see above) of the gene coding in Solanum tuberosum for cinnamyl alcohol dehydrogenase, isoforms 2 (CAD2).
  • SEQ ID NO: 4 corresponds to the translated amino acid sequence of the enzyme.
  • the nucleic acid gene silencing construct comprises at least twice any of the sequences as defined in (a) or (d) when referring to SEQ ID NO: 3, wherein these at least two sequences are disposed in sense and antisense orientations, optionally separated by an intron fragments.
  • the nucleic acid gene silencing construct is an interference RNA coding-DNA that comprises two copies of
  • the nucleic acid gene silencing construct comprises SEQ ID NO: 17.
  • SEQ ID NO: 17 is a sequence of 3943 nucleotides comprising two copies of SEQ ID NO: 3 in sense (from nucleotide 1451 to nucleotide 1732 of SEQ ID NO: 17) and antisense orientation (from nucleotide 2851 to nucleotide 3132 of SEQ ID NO: 17).
  • SEQ ID NO: 17 comprises a nucleotide sequence which is an intron fragment, that comprises a PDK intron (from pyruvate dehydrogenase kinase) and a Cat intron (from catalase).
  • This intron fragment goes from nucleotide 1817 to nucleotide 2794 of SEQ ID NO: 17.
  • a terminator sequence (NOS_T from nopaline synthase) is from nucleotide 3149 to nucleotide 3913 of SEQ ID NO: 17.
  • AttB sequences are present at 5'- and 3'-ends, namely an attB1 from nucleotide 5 to 29 of SEQ ID NO: 17; and an attB2 (in anti-sense orientation) from nucleotide 3915 to nucleotide 3939 of SEQ ID NO: 17. These attB sequences allow the cloning in a
  • Isolated transgenic cells of stems and leaves are particular embodiments of the transgenic plant cells of the third aspect of the invention.
  • tuber or tuber fragments also known as tuber seeds
  • the nucleic acid gene silencing construct comprises a nucleotide sequence of a gene of the Solanum tuberosum coding for an enzyme of the lignin pathway synthesis; or comprises a nucleotide sequence with at least 80 % of identity with said nucleotide sequence of a gene of the
  • Solanum tuberosum coding for an enzyme of the lignin pathway synthesis is sold.
  • nucleic acid gene silencing construct in the cells capable of down-regulating the expression of a gene coding for an enzyme of the lignin biosynthesis pathway, have been disclosed above when referring to the transgenic Solanum genus plant of the first aspect of the invention.
  • a particular embodiment is a mixture of grinded and/or milled leaves and stems, thus a product from the aerial part of the plant.
  • the manufactured plant product is a part of the plant previously submitted to an alcoholic extraction. More particularly, the insoluble residual of stems, leaves or mixtures thereof extracted with an alcoholic solution (i.e. ethanol 70 % in water), which stems, leaves or mixtures thereof can optionally be grinded and/or milled before or after extraction.
  • alcoholic extraction is to be understood a process in which the parts of the plant are contacted, with or without agitation, with an alcoholic solution.
  • Another particular embodiment is a mixture of the aerial part of the plant and the roots and optionally the tuber, which are milled and/or grinded to pellets.
  • the milling of the different parts of the plant leads to powdered solid mixtures of different grain size depending of the mill and the sieve used to separate the solids.
  • the plant or parts of the plant can be triturated and further ground to conform pellets.
  • the pellets (relatively spherical and/or rod-shaped) of the plant of the invention can be advantageously obtained by extruding the triturated parts while warming, since lignin of the plant melts and allows conformation and resistance of the pellet without having to add any other additive, such as a binder.
  • suitable binders may also be used for conformation of the pellets if needed.
  • the manufactured plant product also encompasses any dried or partially dried aerial part of the plant duly packaged in nets after harvesting, for example packaged in nets for baling, commonly used in agricultural applications.
  • the genetically modified plants of the Solanum tuberosum are produced by a method comprising:
  • tuberosum plant with a nucleic acid gene-silencing construct capable of down- regulating the expression of a gene coding for an enzyme of the lignin biosynthesis pathway, to obtain a transformed cell and or tissue;
  • step (a) is carried out with a nucleic acid gene-silencing construct that comprises a nucleotide sequence of a gene of the Solanum tuberosum coding for an enzyme of the lignin pathway synthesis; or comprises a nucleotide sequence with at least 80 % of identity with said nucleotide sequence of a gene of the Solanum tuberosum coding for an enzyme of the lignin pathway synthesis.
  • the genetically modified plants of the Solanum tuberosum of the invention can be produced by down-regulation (also known as silencing) of any or both of the genes coding for an enzyme or combination of enzymes selected from cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase.
  • Solanum tuberosum may be in particular produced by the method comprising:
  • step (b) selecting transformed cells and/or tissues of step (a);
  • step (c) regenerating an entire plant from the transformed cell and/or tissue of step (b).
  • the plant cell and/or plant tissue is selected from the group consisting of a gametophyte, a callus of the plant, an explant of the plant, an isolated cell from leaves, stems, and/or roots, and mixtures of these cells and/or tissues. In a more particular embodiment, it is a leaf explant. In another particular embodiment, optionally in combination with any
  • the nucleic acid gene-silencing construct used for transforming the plant cell and/or tissue comprises a sequence selected from the group consisting of:
  • the nucleic acid gene- silencing construct used for transforming the plant cell and/or tissue comprises a sequence selected from the group consisting of (a) a nucleotide sequence comprising sequence SEQ ID NO: 1 , preferably a nucleotide sequence consisting in SEQ ID NO: 16; (b) a nucleotide sequence comprising sequence SEQ ID NO:3, preferably a nucleotide sequence consisting in SEQ ID NO: 17; and mixtures of these sequences, which means a composition with a combination of both.
  • the nucleic acid gene- silencing construct used for transforming the plant cell and/or tissue is selected from the group consisting of an antisense-DNA, interference RNA coding-DNA, and combinations thereof.
  • interference RNA coding-DNA giving rise to an interference RNA selected from the group consisting of dsRNA, ddRNA and siRNA.
  • interference RNA coding-DNA that comprises a sequence comprising two copies of SEQ ID NO: 1 or of SEQ ID NO: 3, each separated by an intron fragment, and that gives rise to a dsRNA when transcribed. This dsRNA incorporates into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the nucleic acid gene- silencing construct is comprised in a plant transformation vector.
  • Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants.
  • the most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, the common lab bacterium, and
  • Agrobacterium tumefaciens a bacterium used to insert the recombinant (customized) DNA into plants.
  • plant transformation vectors include binary vectors compatible with the Gateway conversion cassettes (Invitrogen), and which facilitate the stable introduction of hairpin RNAi constructs into host plant genomes. With these vectors, foreign DNA (target gene) can be introduced in the cells of the plants in a non-lethal way. The resulting transgenic offspring can be selected using kanamycin, due to the simultaneously introduced NPTII gene.
  • the steps of (b) selecting transformed cells and/or tissues of step (a); and (c) regenerating an entire plant from the transformed cell and/or tissue of step (b) are performed, respectively, by detecting by means of a molecular biology tool if the nucleic acid gene-silencing construct has been entered in the cells and/or tissue of the plant; and by inducing plant growth of the transformed cell/tissue
  • RNA is retro-transcribed to cDNA and this later is detected by means of complementary probes, for example
  • regenerating culture mediums are select from the group consisting of callus induction medium (CIM), shoot induction medium (SIM), and root induction medium (RIM). Plantlets with well-developed roots can then be multiplied in in-vitro culture or transferred to soil.
  • CIM callus induction medium
  • SIM shoot induction medium
  • RIM root induction medium
  • the invention also encompasses a method of producing bioethanol comprising:
  • the glucose is extracted from the aerial part of the Solanum tuberosum plant.
  • the third internode being the internodes those stem parts between nodes and being the first internode the first stem part between nodes starting from the bottom of stem.
  • Extracted glucose may be quantified by any method known in the art.
  • the method of producing bioethanol according to the invention encompasses, in a particular embodiment a pre-treatment step in which the cell wall of the plant is broken to help separating out the lignin.
  • This pre-treatment is in particular performed with mild technologies such as by supercritical water or steam and pressure, and supercritical CO2. These mild technologies avoid the loss of fermentable mass (glucose from cellulose) that is present in other more aggressive pre-treatment steps comprising acid (sulphuric acid) or alkali treatment (ammonia).
  • one of the main goals of the plants of the invention is that due to its lignin content and type of lignin this pre-treatment steps can be suppressed or, if optionally performed there can be avoided aggressive treatments and/or pre-treatment times, thus reducing interfering compounds in further steps and of course reducing global costs.
  • the extraction of glucose is performed
  • Enzyme sources include, but are not limited to cellulose from Trichoderma reseei and cellobiase from Aspergillus niger.
  • the method of producing bioethanol using enzyme mixtures in step (a) and those of the fermentation in step (b) is commonly known for the skilled man as the enzymatic hydrolysis of biomass (plant cell wall).
  • fermenting the extracted glucose to bioethanol may be performed by means of a mixture of fermentation microorganisms, most particularly fermentation yeast.
  • These microorganisms for performing step (b) convert, in a particular embodiment, the extracted glucose to ethanol in a one-pot reaction while glucose from cellulose is being extracted from step (a).
  • step (b) it further comprises the step of distilling the bioethanol obtained in step (b) to remove water.
  • Example 1 Generation of RNAi constructs and Generation of transgenic potato plants
  • RNA interference (RNAi) for gene silencing was chosen.
  • silencing cassettes for CCR1 and CAD2 were entrusted to GeneArt® Gene Synthesis (Life Technologies). The silencing cassettes consisted, respectively in SEQ ID NO: 16 and 17 (depicted in Table 5).
  • Each of the cassettes contained the Cauliflower Mosaic Virus 35 S promoter (CaMV 35S), the sense gene fragment, the two introns from PDK (from pyruvate dehydrogenase kinase) and Cat (from catalase), the antisense gene fragment, and the terminator.
  • CaMV 35S Cauliflower Mosaic Virus 35 S promoter
  • the sense gene fragment the two introns from PDK (from pyruvate dehydrogenase kinase) and Cat (from catalase)
  • the antisense gene fragment and the terminator.
  • the 5'- and 3'-ends of the sequences attB1 and attB2 site were included for making them compatible with the commercial Gateway® cloning System (Invitrogen Corp.).
  • Synthetic DNA fragments were introduced into the suitable pDONR Gateway vector pDONR221 by carrying out BP reactions (BP ClonaseTM II enzyme mix) for creating Entry clones, which were finally used to introduce the silencing cassettes into a Gateway compatible binary vector.
  • BP reactions BP ClonaseTM II enzyme mix
  • the silencing constructs were mobilized and transferred into the binary vector called pSP-DEST1 in the manner disclosed by Hentrich et al., in "The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and
  • YUCCA9 gene expression The Plant Journal-2013, vol. no. 74, pp. 626-637.
  • This vector is an offspring of pGTV-BAR (Becker et al., "Binary vectors which allow the exchange of plant selectable markers and reporter genes", Oxford University Press 1990 Nucleic Acids Research, vol. no.: 18(1 ), pp.: 203).
  • Gateway conversion cassette Invitrogen
  • the final silencing construct for each individual gene generated an RNA fragment spanning the gene-specific probe in opposite orientations separated by a non-coding intron region as depicted in FIG. 2 (A and B).
  • PCR was used to verify that the collection of generated pSP-DEST1 vectors contained the expected fragments in the proper orientations (data not shown).
  • a transformation control vector was produced and it was used to assess transformation efficiency and suitability of the approach.
  • the PHYTOENE DESATURASE (PDS) gene of SEQ ID NO: 5 (corresponding to entry PGSC0003DMP400016140 of database Phytozome (version 9.1 )) was selected. Silencing of the PDS gene is supposed to results in a clearly identifiable albino and dwarf phenotype.
  • the vector pSP-DEST1 construct with specific sequences for the potato PDS gene indeed, produced albino plants that were properly detected among transformants.
  • the transformation of potato is a multi-step procedure that involves in-vitro propagation of sterile plant material, Agrobacterium-mediated infection of explants, selection of transformed calli under appropriate selection pressure, and the micropropagation of the calli to regenerate complete transgenic plants, which is accomplished by changing the composition (plant hormone content) of the media to either trigger root or shoot formation.
  • pSP-DEST1 silencing plasmids comprising two copies in both orientations (sense and antisense) of SEQ ID NO:1 or of SEQ ID NO:3 separated by the PDK intron (from pyruvate dehydrogenase kinase) and Cat (from catalase) intron of the plasmid were amplified in E.coli and purified for further transfer into Agrobacterium and plant transformation. To do this, leaves excised from 4-week old in-vitro maintained shoot cultures were used as explants for transformation. To prepare the explants, the petiole and 3 ⁇ 4 of the leave in immediate proximity to the petiole were discarded.
  • Explant size must be approximately of 1 cm 2 . Twenty explants were placed in a Petri dish with 20 ml_ of MS liquid medium (Murashige and Skoog (MS) salts and vitamins, 20 g L “1 sucrose, 20 g L "1 , 0.5 g L "1 2-(N-morpholino)ethanesulfonic acid
  • MES MES
  • explants were dried on filter paper and cultured on callus induction medium (CIM; 4.4 g L “1 MS salts, 16 g L “1 glucose, 0.5 g L “1 MES, 0.1 mg L “1 6-benzylaminopurine (BAP), 5 mg L “1 a-naphthaleneacetic acid (NAA), 7 g L “1 agar, 50 mg L “1 kanamycin, 250 mg L “1 cefotaxime) for 7 days.
  • CCM callus induction medium
  • explants were transferred to shoot induction medium (SIM; 4.4 g L “1 MS salts, 16 g L “1 glucose, 0.5 g L “1 MES, 0.02 mg L “1 a- naphthaleneacetic acid (NAA), 0.02 mg L “1 gibberellic acid A 3 (GA 3 ), 2 mg L “1 zeatin riboside, 7 g L “1 agar, 50 mg L “1 kanamycin, 250 mg L “1 cefotaxime).
  • shoot induction medium SIM; 4.4 g L “1 MS salts, 16 g L “1 glucose, 0.5 g L “1 MES, 0.02 mg L “1 a- naphthaleneacetic acid (NAA), 0.02 mg L “1 gibberellic acid A 3 (GA 3 ), 2 mg L “1 zeatin riboside, 7 g L “1 agar, 50 mg L “1 kanamycin, 250 mg L “1 cefotaxime).
  • protocol of Ohate-Sanchez consisted in grounding frozen plant tissue to a fine powder in liquid nitrogen. A total amount of 20-30 mg were further homogenized in 300 ⁇ cell lysis solution. After an incubation of 5 min at room temperature, 100 ⁇ of protein-DNA precipitation solution was added to the lysate. The tubes were gently inverted and incubated at 4°C for, at least, 10 min. Subsequently, the cell lysate was centrifuged (10 min, 4°C) and the supernatant transferred to fresh reaction tubes. Next, 300 ⁇ isopropanol were added, samples were again centrifuged (4 min), and the resulting supernatant carefully discarded. The pellet was washed with 70% ethanol (v/v), air-dried 5 and resuspended in 25 ⁇ DEPC-water.
  • cDNA was synthesized from 2 g of DNA-free RNA using the avian myeloblastosis virus reverse transcriptase and oligo-(dT)15 primers
  • PDK and Cat are intronic regions present in the constructs.
  • PDK.as means reverse primer for anstisense orientation of target gene.
  • S means sense and as means antisense orinetation of the target gene (either CAD2 or CCR1).
  • the silencing level of the target genes in RNAi plants was determined by RT- qPCR.
  • a LightCycler®480 System (Roche) was used for real-time PCR (10 s at 95°, 45 cycles of 95°C for 10 s, 60°C for 20 s min and 72°C for 30 s) using LightCycler® 480 SYBR Green I Master (Roche).
  • the UBIQUITIN gene (herein termed UBI) from S. tuberosum (corresponding to entry
  • Table 2 shows the primer pairs used for RT-qPCR amplification and the expected size of amplicons.
  • transgenic lines showing reduced expression of the target genes were obtained, though at very different levels, for all the constructs used.
  • CAD2 gene seven lines were analysed, and three of them (2, 4 and 5) displayed very low levels of the target gene expression, whereas four (1 , 3, 8 and 9) showed reduction to medium levels (FIG. 3 (A)).
  • plants with lower expression of the endogenous potato CAD2 gene also exhibit a characteristic red colour in stems as a consequence of alterations in lignin content and the resulting accumulation of monolignol precursors such as ferulic acid.
  • Data from relative expression are depicted for each gene (CAD2 and CCR1) in following Table 3.
  • FIG. 4 shows two pictures of the transgenic plants.
  • FIG. 4(A) illustrates line 2 (L2) of the plants down- regulated for CAD2 gene
  • FIG. 4(B) illustrates line 7 (L7) of the plants down-regulated for CCR1 gene.
  • FIG. 5 illustrates several pictures with the tubers of plants of lines 9 (L9) and 4 (L4) down-regulated for CAD2 gene; and with the tubers of line 8 (L8) of down-regulated for CCR1 gene.
  • WT wild-type tuber
  • transgenics were subjected to closer inspection of their cell wall properties, including the analysis of total lignin contents, glucose extractability, and their monolignol ratios. In the following the obtained results will be presented.
  • Total lignin content was determined for each of the five independent transgenic lines selected for each silenced gene.
  • the total lignin content of a given sample represents an important value that should be assessed alongside with the lignin composition.
  • the sample had to be vortexed thoroughly, followed by centrifugation at 14,000 rpm for 10 min to pellet the alcohol insoluble residue containing the cell wall material. The supernatant had to be carefully removed by aspiration, paying attention not to disturb the pellet.
  • the pellet was washed in 1 ml_ of chloroform/methanol (1 :1 v/v) by thoroughly vortexing the tube to resuspend the pellet. The sample was again centrifuged at 14,000rpm for 10 min and the supernatant was carefully removed by aspiration, without disturbing the resulting pellet. Finally, the sample was taken to dryness using a vacuum concentrator. Prepared samples were stored at -80 °C or on dry ice until they were further processed
  • thioacidolysis was performed for the analysis of lignin composition. The quantitative comparison of monolignol compositions among plant samples from wild type and knockdown lines was determined.
  • lignin has to be chemically decomposed into its monomeric building blocks by a chemical process, referred to as thioacidolysis.
  • thioacidolysis For each sample, 2 mg of cell wall material (processed as above indicated) was weighed into screw capped glass tubes for thioacidolysis. Chemical decomposition of the contained lignin was initiated by adding 200 ⁇ of a mixture of
  • phenylpropanoids contained in the preparations described above were derivatized by adding 500 ⁇ of ethyl acetate, 20 ⁇ of pyridine, and 100 ⁇ of
  • N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA+1 % TMCS).
  • the samples were incubated at 60°C for 1 h. After the incubation time 100 ⁇ of the mixture were transferred to GC-vials and, if necessary, 100 ⁇ of hexane added.
  • One ⁇ of the derivatized sample was injected splitless by an CLC CombiPal autosampler into a BRUKER Daltonics 451 gas chromatograph equipped with a 30 m x 0.25 mm i.d. fused silica capillary column with a chemically bonded 0.25 ⁇ ZB 5ms stationary phase (Phenomenex).
  • the injector temperature was set to 250°C.
  • the split was opened (1 :100).
  • a pressure pulse (30 psi) supported sample application onto the column.
  • the gas flow rate through the column was adjusted to 1 ml/min, the column temperature was held at 50°C for 1 .20 min, increased by 30°C/min to 120°C, then increased by 10°C/min to 325°C, and held there for 5 min.
  • the column effluent was introduced into the ion source of a Scion-TQ triple quadrupole mass spectrometer, GC-QQQ-MS (BRUKER Daltonics).
  • the transfer line and the ion source temperatures were maintained at 250°C. Ions were generated by a 70 eV electron beam at an ionization current of 80 ⁇ , and 30 spectra/s were recorded in the mass range 50 to 500 m/z.
  • Diagnostic electron impact (El) mass spectra could be obtained for all three monolignols. Retention times of 12.98 min, 14.42 min, and 15.82 min could be determined for p-coumaryl alcohol (H), coniferyl alcohol (G) and sinapyl alcohol (S), respectively Advancing the sensitivity of the described EI-MS method initially used to identify the specific retention times for H-, G-, and S-phenylpropanoid units, it was developed a more comprehensive method that facilitates more sensitive quantification of the three monolignols by employing a multiple reaction monitoring MS protocol.
  • H p-coumaryl alcohol
  • G coniferyl alcohol
  • S sinapyl alcohol
  • FIG. 6 indicates, respectively, the lignin content (FIG. 6 (A)), glucose extractability (FIG. 6 (B)) and the monolignol ratios (FIG. 6 (C)) in the assayed lines (L2, L5, L8 and L9) of the transgenic plant in which CAD2 gene has been down-regulated (silenced at several levels in relation to WT, not comprising the nucleic acid gene silencing construct) Monolignol ratios of the transgenic plants are also deducible from values illustrated in next Table A.
  • Table A the lignin content
  • FIG. 6 (B) glucose extractability
  • FIG. 6 (C) monolignol ratios
  • the X-axis indicates the particular assayed genotype (G) or lines (L2, L5, L8, L9).
  • G the lignin content is expressed as g lignin/mg cell wall.
  • glucose extractability is indicated as picomols (pmol) of glucose/mg cell wall.
  • C the Y-axis is the relative abundance of H lignin (first left bar in each line L), G lignin (second bar in the middle of each line L), and S lignin (third right bar in each line L).
  • FIG. 6 From this FIG. 6 is can be concluded that in comparison to wild-type internodes of potato variant Desiree, the CAD2RNAi lines L8 and L9 showed significantly reduced total lignin contents, while the other inspected lines do not show substantial alterations.
  • the CAD2RNAi lines namely lines L2, L5, L8, and L9, showed a drastically improved extractability of glucose.
  • the lines L8 and L9 showed an improvement of 7.65- and 8.46-fold, respectively.
  • the inventors proposed that a reason for this improvement could be the significant shift in monolignol ratios that occurred in consequence of the genetic manipulation made.
  • lines L2, L8, and L9 show a considerable shift from S- to H-lignin, whereas L5 feature a shift from S- to G-lignin.
  • FIG. 7 shows respectively, the lignin content (FIG. 7 (A)), glucose extractability (FIG. 7 (B)) and the monolignol ratios (FIG. 7 (C)) in the assayed lines (L1 , L2, L3, L7 and L8).
  • the aerial part of the plants of some transgenic lines (plant without tubers and roots) and of the wild-type were weighed before and after drying.
  • the dry weight was obtained by taking the plant to dryness (0 %- 3% humidity) using heat (thermo incubator at 55 °C). Drying was monitored over 6 days.
  • Clause 1 - Genetically modified plant of the Solanum genus comprising a lignin composition with a ratio of the amounts of the monolignols sinapyl alcohol (S) /coniferyl alcohol (G) in the cell wall from 0.10 to 0.50, each monolignol amount expressed as picomols of monolignol/milligrams of cell wall.
  • Clause 3. The plant according to any of clauses 1 -2, wherein the S/G ratio in the cell wall is from 0.25 to 0.35, each monolignol amount expressed as pmols of monolignol/mg of cell wall.
  • nucleic acid gene silencing construct is selected from the group consisting of an antisense-DNA, an interference RNA coding-DNA, and combinations thereof.
  • nucleic acid gene silencing construct is an interference RNA coding-DNA.
  • Clause 7. The plant according to any of clauses 4-6, wherein the enzyme is cinnamoyl CoA reductase and the nucleic acid gene silencing construct comprises a sequence selected from the group consisting of:
  • nucleic acid gene silencing construct comprises SEQ ID NO: 16.
  • Clause 9. The plant according to any of clauses 4-6, wherein the enzyme is cinnamyl alcohol dehydrogenase and the nucleic acid gene silencing construct comprises a sequence selected from the group consisting of:
  • nucleic acid gene- silencing construct comprises SEQ ID NO: 17.
  • Clause 1 1 A part of a plant as defined in any of clauses 1 -10 selected from the group consisting of a seed, a leaf, a stem, a tuber, a fruit and a root.
  • Clause 13. A manufactured plant product of the plant according to any of clauses 1 -10, or alternatively of a part of the plant as defined in clause 1 1 , said manufactured plant product selected from the group consisting of milled parts of the plant, ground parts of the plant, and packaged parts of the entire aerial part of the plant.
  • Clause 14. A method of producing the plant according to any of clauses 1 -10, comprising:

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Abstract

L'invention concerne des plantes génétiquement modifiées du genre Solanum comprenant une composition de lignine dotée d'un rapport particulier entre les quantités des monolignols alcool synapylique (S)/alcool coniférylique (G) dans la paroi cellulaire, qui permettent une meilleure capacité d'extraction de glucose sans compromettre l'intégrité de la plante. L'invention concerne également des parties, des cellules et des produits végétaux manufacturés, dérivés de ces plantes génétiquement modifiées. Des procédés particuliers pour obtenir les plantes génétiquement modifiées comprenant la régulation négative des enzymes CCR1 et CAD2 sont également décrits, ainsi que des procédés de fabrication de bioéthanol à partir desdites plantes génétiquement modifiées.
PCT/EP2016/053541 2015-02-20 2016-02-19 Plantes de solanum tuberosum pour la production de biocarburant WO2016131951A1 (fr)

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Citations (3)

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WO2008064289A2 (fr) 2006-11-21 2008-05-29 The Samuel Roberts Noble Foundation, Inc. Procédés et compositions de production de biocarburant
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WO2008069964A2 (fr) * 2006-12-01 2008-06-12 The Board Of Trustees Of Michigan State University Modification de la régulation d'enzymes de biosynthèse de la lignine du maïs au moyen d'une technologie basée sur l'arni
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