WO2013005211A2 - Matériels végétaux de complexation du bore et leurs utilisations, en référence croisée à des applications connexes - Google Patents

Matériels végétaux de complexation du bore et leurs utilisations, en référence croisée à des applications connexes Download PDF

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WO2013005211A2
WO2013005211A2 PCT/IL2012/050233 IL2012050233W WO2013005211A2 WO 2013005211 A2 WO2013005211 A2 WO 2013005211A2 IL 2012050233 W IL2012050233 W IL 2012050233W WO 2013005211 A2 WO2013005211 A2 WO 2013005211A2
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Prior art keywords
plant
boron
fructose
combination
glucose
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PCT/IL2012/050233
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WO2013005211A3 (fr
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Menachem Moshelion
Nava MORAN
Netta Li LAMDAN
Ziv ATTIA
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2013005211A2 publication Critical patent/WO2013005211A2/fr
Publication of WO2013005211A3 publication Critical patent/WO2013005211A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/28—Treatment of water, waste water, or sewage by sorption
    • C02F1/286—Treatment of water, waste water, or sewage by sorption using natural organic sorbents or derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
    • C02F3/327—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae characterised by animals and 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
    • 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
    • C12N15/8259—Phytoremediation
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/66—Treatment of water, waste water, or sewage by neutralisation; pH adjustment
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/108—Boron compounds
    • 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
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • This invention relates to the field of botany and more specifically to plant materials, the reduction of the concentration of boron in water via the use of same, in particular in desalinated water and other uses thereof.
  • Boron (B) is a small, water soluble molecule, found in sea water at concentrations of 3-6 mg/L (3-6 ppm, or, roughly, 0.3-0.6 mM). Elsewhere, in the ground water and in the soil, boron concentrations vary widely and depend on the surrounding geology and wastewater discharges.
  • B Boron
  • B(OH) 3 boric acid
  • boron in the form of boric acid (pKa 9.24) and ⁇ 0.05% is in the form of borate (tetrahydroxyborate: B(OH) 4 " ) ions.
  • B(OH) 4 " tetrahydroxyborate
  • cytoplasm at pH about 7.5
  • more than 98% of boron is in the form of boric acid and less than 2% in the form of borate ion.
  • boron complexation with two molecules of alcoholic sugars e.g. fructose, sorbitol, mannitol, dulcitol
  • a pair of cis-hydroxyls has been suggested to impart mobility to boron in the phloem, allowing this generally stationary element to move to sinks.
  • Boron complexation with cis-hydroxyls has been also suggested as a mechanism of averting boron toxicity (i.e., as a mechanism of cellular boron tolerance) in some Brassicaceae.
  • boron is already toxic to arthropods and to some plants and diminishes agricultural crops.
  • boron-sensitive plants 0.5-1 mg boron/ L, i.e., 0.05-0.1 mM
  • blackberry citrus, peach, cherry, plum, grape, cowpea, onion, garlic, sweet, potato, wheat, barley, sunflower, sesame, strawberry.
  • a visible symptom of boron toxicity in a variety of plant species is necrosis along leaf margins.
  • boron is mobile in the phloem toxicity symptoms may rather occur in the fruit (such as gummy nuts and internal necrosis) and in other sink tissues (such as necroses in buds and young stems).
  • boron toxicity is evident as decreased fruit yield of crops (for example, tomato fruit yield decreased by 3.7% with each additional increase of 0.1 mM B in the soil solution above a threshold of 0.53 mM B).
  • Physiological effects of boron toxicity include reduction in cell division in the root, reduction in elongation rates of the root and the shoot, decreased chlorophyll level in the leaf, reduction in photosynthesis and stomatal conductance, excess production of lignin and suberin, decreasing of external root zone acidification, increase of membrane leakiness and increased fatty acids oxidation.
  • Some plants such as various crop cultivars or halophytes, are significantly more resistant to boron than other members of their family but the mechanism whereby such plants are resistant to boron is unknown.
  • this invention further provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water.
  • this invention further provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water
  • reference to "increased concentration of malic acid, fructose, citric acid or a combination thereof” refers to a concentration that is higher than that present in a plant member of a related plant family.
  • such methods referring to an "increased concentration of malic acid, fructose, citric acid or a combination thereof" refer to a concentration of malic acid, fructose and glucose ranging from at least 2 mmol kg "1 FW to 20 mmol kg ln
  • the plant material is fresh or dried.
  • the plant material is derived from a Thellungiella salsuginea plant, which is also known in the art as being a Thellungiella halophila plant.
  • the water is contacted with a solid support comprising said plant material and in some embodiments, the solid support includes a column or other filtering device.
  • a plant exhibiting increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof is grown by hydroponics within the water, as part of such method.
  • this invention provides a solid support comprising a plant material exhibiting an increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member, for a period of time sufficient to promote boron accumulation within said plant material.
  • the plant material is derived from a Thellungiella salsuginea plant.
  • the solid support is a column or filtering device.
  • this invention provides for a method for reducing the boron concentration in soil, said method comprising contacting soil containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member, for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said soil.
  • this invention further provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby said plant exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family member for a period of time sufficient to promote boron accumulation within said plant material.
  • such methods further comprise the step of engineering the plant to express one or more genes in an altered manner, such that altered expression of said one or more genes results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof.
  • this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron reducing toxicity of boron to said plant, thereby being a method of increasing boron tolerance in said plant.
  • such method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms said intracellular complexes.
  • the plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor ⁇ family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.
  • CBF3 Core Binding Factor ⁇ family-3
  • DHAR dehydroascorbate reductase
  • the gene is from an alternate plant species than that of said plant, or in some embodiments, the gene is bacterial in origin. In some embodiments, the expression of the gene is inducible.
  • the invention provides a plant engineered in accordance with the methods of this invention and in some embodiments, the invention provides a seed of a plant or a transgenic plant thus engineered.
  • the plant or seed is of a plant comprising a crop, flowering plant, grainbearing plant, fruitbearing plant, nutbearing plant, herb, turf grass, sod or seedling.
  • the method further comprises the step of engineering the plant to under-express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.
  • this invention provides a method for reducing soil boron content, the method comprising:
  • the method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron and such an engineered plant is then grown in the soil.
  • the soil is irrigated with desalinated water.
  • the soil bounds or lines a water reservoir.
  • Figure 1 depicts hydroponic growth of Arabidopsis and Thellungiella.
  • Figure 1A presents a top view, plantlets growing in rockwool-filled test tubes.
  • Figure IB depicts a root system hanging down from the 4 cm test tubes (lifted out of the aerated nutrient solution for photography and exposing the red air-stones). For measurements the roots were gently pried separate while in water.
  • Figure 2 shows the effect of boron on Arabidopsis and Thellungiella grown in soil.
  • Figure 2A depicts thaliana and T. halofila plants watered for 23 days with solutions containing boric acid at the indicated concentrations. The photos were taken on day 23 after start of boron exposure. Note the relative tolerance of Thellungiella to boron. Similar effects were observed in at least five independent experiments.
  • Figure 2B graphically depicts the concentration of accumulated boron, [B]int, in the shoot of plants harvested on day 23 of exposure as a function of boron concentration in the irrigation solution, [B]ext, expressed as mmol per kg of fresh weight, FW, of the whole shoot.
  • Figure 3 depicts the effect of boron on Arabidopsis and Thellungiella grown in a hydroponic system and exposed for 7 days to boric acid at the indicated concentrations in the growth solution. Photos taken on day 7. Note the relative tolerance of Thellungiella to boron. Similar behavior was observed in at least five independent experiments.
  • Figure 4 depicts the concentration of plant-accumulated boron, [B]int, as a function of boron concentration in the root-bathing solution, [B]ext.
  • Figure 4A graphically depicts the accumulation in the root. [B]int is expressed in mM boron in the root "water” (FW-DW).
  • FIG. 4D depicts the mean transpiration ( ⁇ SE) determined in three (Control) or two (B(5)) independent experiments from the indicated number of pots (each with 2-4 plants; see detailed description of measurement below). 5 days of boron treatment did not affect transpiration, in agreement with other experiments with soil grown plants, where 5 days of B treatment did not affect plant appearance. In parallel, in control conditions, Thellungiella transpired on average about 50 % more than Arabidopsis (*: p ⁇ 0.02; 2-tail t test).
  • Figure 4E depicts an independent experiment similar to Figure 4D.
  • Figure 4F shows a scan of leaves (table-top office scanner HP7130) of plants from one pot.
  • Figure 4G shows the conversion to contours and calculation of the enclosed area as preformed by ImageJ software (by W. Rasband, NIH, USA, http://rsb.info.nih.gov/ij).
  • ImageJ software by W. Rasband, NIH, USA, http://rsb.info.nih.gov/ij.
  • the mean FW of a unit leaf area was similar in soil-grown Arabidopsis and Thellungiella (22.5 ⁇ 1.8 ( ⁇ SE) and 21.5 ⁇ 0.2 mg.cm-2, respectively, obtained by dividing the WF of the shoot by the surface area of its leaves.
  • Figure 6 depicts the abundance of polyol metabolites in the shoots of Thellungiella and Arabidopsis grown in hydroponics.
  • the mean concentrations of the metabolites were higher in Thellungiella than in Arabidopsis under the same treatments (*: p ⁇ 0.02, **: p ⁇ 0.01, ***: p ⁇ 0.001; see also Table 2, Expts. Ill, IV).
  • the mean RATIO in B-treated Thellungiella (31) was significantly larger than the mean RATIO in Arabidopsis (0.72) (*: p ⁇ 0.05, one -tailed t test); but because of the great variability of the individual values both mean RATIOs Ratios differed from '2' only at a low significance level (Is: p ⁇ 0.1; single-tail t test). Note the break in the ordinate. These data appear also in Table 4.
  • This invention provides, in some embodiments, for the increased accumulation of boron within plant materials and use of the same for boron removal from water and soil sources. Related applications of the same are described herein, as well.
  • increased boron accumulation within plant materials is effected by providing conditions such that an increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof is effected in plant shoots of interest, which malic acid, fructose, glucose, citric acid or a combination thereof are available for complexation with boron, providing an intracellular boron detoxification process in such plants.
  • conference of boron tolerance to plant materials is a contemplated result of the same.
  • Example 4 describes the harnessing of this finding to provide materials and methods for the reduction of boron concentration in water, in a manner that is commercially meaningful.
  • the term "increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof” refers to a plant material exhibiting increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at concentrations ranging from 2 mmol kg "1 fresh weight (FW) to 20 mmol kg "1 FW.
  • the term "increased accumulation of malic acid, fructose, glucose, citric acid or a combination thereof” refers to plant material exhibiting increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration in comparison to the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in plants of related family members, etc.
  • this invention provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a plant material, wherein said plant material exhibits an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants for a period of time sufficient to promote boron accumulation within said plant material and reduction of boron concentration in said water.
  • such method and materials for use in accordance with such purpose is to reduce the boron concentration in water sources, which sources are derived from desalinated water, industrially contaminated water, or any other appropriate water source, whereby such water source contains an undesirable concentration of boron, whose reduction is desired.
  • the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect is fresh or dried.
  • the plant material for use in accordance with the methods as herein described is ground, chopped or further processed.
  • plant material refers inter alia, to any element of a plant which is useful in accordance with the methods as herein described.
  • plant material refers to whole plants, cuttings of plants, shoots, leaves, and plant extracts and combinations thereof.
  • plant material specifically includes materials derived from a Thellungiella salsuginea plant.
  • the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect is derived from a Thellungiella salsuginea plant.
  • the water is contacted with a solid support comprising said plant material, and in some embodiments, such solid support includes a column or filtering device.
  • the plant material for use in accordance with the methods and for incorporation within the materials according to this aspect exhibit increased concentrations of malic acid, fructose, glucose, citric acid or a combination thereof and is grown by hydroponics within said water.
  • the plant material is derived from a plant engineered to over-express one or more genes, whose over-expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron.
  • the plant material is derived from a plant engineered to over-express phosphoenol pyruvate carboxylase, Core Binding Factor ⁇ family-3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof.
  • CBF3 Core Binding Factor ⁇ family-3
  • DHAR dehydroascorbate reductase
  • the plant material is derived from a plant engineered to under-express or fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof which in turn forms intracellular complexes with said boron.
  • the method further includes a step to increase the production of malic acid, fructose, glucose or citric acid within Thellungiella or other plant materials as herein described by growing the same in high salt conditions.
  • the invention provides a solid support comprising a plant material exhibiting an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof.
  • the solid support comprises a plant material exhibiting an increased concentration of malic acid, fructose, glucose, citric acid or a combination thereof at a concentration that is greater than that the concentration of malic acid, fructose, glucose, citric acid or a combination thereof in wild type Arabidopsis plants, or in a plant of a related family, etc.
  • the plant material is fresh or dried.
  • the plant material is derived from a Thellungiella salsuginea plant.
  • the solid support is a column or filtering device.
  • a column as envisioned herein may be prepared by any conventional means, as will be appreciated by the skilled artisan.
  • the column may be packed by mixing crushed plant material with sand in various proportions and packing the column with the same.
  • the granule size may be adjusted to manipulate the column resistance to flow.
  • a porous fused silica glass serves as an exit filter.
  • this invention provides a method for reducing the boron concentration in water, said method comprising contacting water containing an undesirable concentration of boron with a brown algae material for a period of time sufficient to promote boron accumulation within said brown algae material and reduction of boron concentration in said water.
  • this invention provides a solid support comprising a brown algae material, which complexes with boron.
  • the brown algae is Saragassum vulgaris.
  • this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby said plant exhibits increased shoot concentrations of malic acid, fructose, glucose, citric acid or a combination thereof.
  • such methods further comprise the step of engineering the plant to express one or more genes in an altered manner, such that altered expression of said one or more genes results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof.
  • metabolic pathways can be biased by overexpression of certain genes, by knockout/downmodulated expression of some genes and in some cases by combinations thereof to result in an accumulation or enhanced production of a desired product of such pathway.
  • this invention provides a method for increasing boron tolerance in a plant said method comprising providing conditions whereby a shoot in said plant contains intracellular complexes of malic acid, fructose, glucose, citric acid or a combination thereof with boron reducing toxicity of boron to said plant, thereby being a method of increasing boron tolerance in said plant.
  • such method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms said intracellular complexes.
  • the plant is engineered to overexpress phosphoenol pyruvate carboxylase, Core Binding Factor ⁇ family -3 (CBF3) or dehydroascorbate reductase (DHAR) or a combination thereof [.
  • CBF3 Core Binding Factor ⁇ family -3
  • DHAR dehydroascorbate reductase
  • such methods make use of a vector construct comprising a nucleic acid encoding the gene of interest.
  • the method is further effected by producing a plant comprising the vector, wherein the plant exhibits enhanced boron tolerance.
  • Genes of interest intended for expression in plants are first assembled in expression cassettes comprising a promoter.
  • Methods which are well known to or developed by those skilled in the art, may be used to construct expression vectors containing such gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Exemplary techniques are widely described in the art (see e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., herein incorporated by reference).
  • these vectors comprise a nucleic acid sequence encoding the gene of interest, operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.
  • promoter e.g., promoters, polyadenylation signals, etc.
  • Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmental-specific promoters, and inducible promoters.
  • Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase ("LAP,” Chao et al., Plant Physiol 120: 979-992 (1999), herein incorporated by reference); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzofhiadiazole-7- carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (e.g.
  • U.S. Pat. No. 5,187,267, herein incorporated by reference a tetracycline-inducible promoter (e.g. U.S. Pat. No. 5,057,422, herein incorporated by reference); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al, EMBO J. 4: 3047-3053 (1985), herein incorporated by reference).
  • seed-specific promoters such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al, EMBO J. 4: 3047-3053 (1985), herein incorporated by reference).
  • the expression cassettes may further comprise any sequences required for expression of mPvNA.
  • sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • transcriptional terminators are available for use in expression of sequences using the promoters of the present invention.
  • Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation.
  • Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (see e.g., Odell et al., Nature 313:810 (1985); Rosenberg et al., Gene 56: 125 (1987); Guerineau et al, Mol. Gen. Genet.
  • constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants.
  • Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Callis et al., Genes Develop. 1 : 1183 (1987), herein incorporated by reference). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
  • the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Kalderon et al, Cell 39:499 (1984); Lassner et al, Plant Molecular Biology 17:229 (1991)), a plant translational consensus sequence (Joshi, Nucleic Acids Research 15:6643 (1987)), an intron (Luehrsen and Walbot, MolGen Genet. 225:81 (1991)), and the like, operably linked to the nucleic acid sequence encoding a gene of interest.
  • a regulator such as a nuclear localization signal (Kalderon et al, Cell 39:499 (1984); Lassner et al, Plant Molecular Biology 17:229 (1991)), a plant translational consensus sequence (Joshi, Nucleic Acids Research 15:6643 (1987)), an intron (Luehrsen and Walbot, MolGen Genet. 225:81 (1991)), and the like, operably linked to the nucleic
  • various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation and, as appropriate, in the desired reading frame.
  • adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
  • transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259 (1982); Bevan et al., Nature 304: 184 (1983), all of which are incorporated herein by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res. 18: 1062 (1990); Spenceret al., Theor. Appl. Genet.
  • the (Ti (T-DNA) plasmid) vector is adapted for use in an Agrobacterium mediated transfection process (see e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are herein incorporated by reference).
  • strains of Agrobacterium tumefaciens are C58, LBA4404, EHA101, C58ClRifR, EHA105, and the like. Examples of Agrobacterium mediated transfection in turfgrasses are provided in PCT Patents WO00/04133; WO00/11138; and U.S. patent application Pub. Nos. 20030106108 Al ; 20040010816A1 ; and U.S. Pat. No. 6,646,185; all of which are herein incorporated by reference.
  • the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJl shuttle vector and the non-oncogenic Ti plasmid pGV3850.
  • T- DNA as a flanking region in a construct for integration into a Ti- or Ri-plasmid has been described in EPO No. 116,718 and PCT Appln. Nos. WO 84/02913, 02919 and 02920 all of which are herein incorporated by reference).
  • the second system is called the "binary" system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation.
  • the other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available.
  • the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium- derived sequences.
  • plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (e.g U.S. Patent No., 5,501,967, herein incorporated by reference).
  • T-DNA Agrobacterium transfer-DNA
  • Homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene.
  • Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.
  • nucleic acids comprising the gene of interest are utilized to construct vectors derived from plant (+) RNA viruses (e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof).
  • RNA viruses e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof.
  • the inserted polynucleotide can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or other promoter.
  • fusion protein e.g., coat protein fusion protein
  • Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785; all of which are incorporated herein by reference.
  • the nucleic acid sequence of interest is introduced directly into a plant.
  • One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (e.g. WO 93/07278; herein incorporated by reference).
  • a nucleic acid sequence encoding a gene of interest is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation.
  • the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.
  • direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; and PCT Patent WO 95/16783; all of which are incorporated herein by reference).
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistic or protoplast transformation with calcium chloride or PEG).
  • the 1 to 1.5 kb flanking regions facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • targeting sequences Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., PNAS, 87: 8526-8530 (1990); Staub and Maliga, Plant Cell, 4: 39-45 (1992), all of which are incorporated herein by reference).
  • vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (e.g. Crossway, Mol. Gen. Genet, 202:179(1985)).
  • the vector is transferred into the plant cell by using polyethylene glycol ((e.g. Krens et al., Nature, 296:72 (1982); Crossway et al., BioTechniques, 4:320 (1986)); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (e.g.
  • the vector may also be introduced into the plant cells by electroporation (e.g. Fromm, et al., Proc. Natl. Acad. Sci. USA, September;82(17):5824-5828 (1985) and Nature February 27-March 5;319(6056):791-793 (1986); Riggs and Bates Proc. Natl. Acad. Sci. USA August;83(15):5602-5606 (1986)).
  • plant protoplasts are electroporated in the -presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
  • the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.) (see e.g., U.S. Pat. No. 4,945,050; and McCabe et al, Biotechnology 6:923 (1988); Weissinger et al., Annual Rev. Genet. 22:421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Svab et al., Proc. Natl. Acad. Sci.
  • devices e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.
  • devices e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.
  • devices e.g., available from Agracetus, Inc., Madison, Wis. and Dupon
  • EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology, 11 : 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat); Wan et al., Plant Physiol, 104: 37 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89:525 (1994) (barley); Knudsen and Muller, Planta, 185:330 (1991) (barley); Umbeck et al., Bio/Technology 5:263 (1987) (cotton); Casas et al., Proc. Natl. Acad. Sci.
  • the vectors comprising a nucleic acid sequence comprising the gene of interest are transferred using Agrobacterium- mediated transformation (Hinchee et al., Biotechnology, 6:915 (1988); Ishida et al., Nature Biotechnology June; 14(6):745-50 (1996), all of which are herein incorporated by reference).
  • Agrobacterium- mediated transformation Hapanee et al., Biotechnology, 6:915 (1988); Ishida et al., Nature Biotechnology June; 14(6):745-50 (1996), all of which are herein incorporated by reference.
  • Heterologous genetic sequences e.g., nucleic acid sequences operatively linked to a promoter of the present invention
  • the Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell, Science, 237: 1176 (1987)). Species, which are susceptible infection by Agrobacterium, may be transformed in vitro.
  • embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
  • Transgenic lines can be established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences comprising the gene of interest may be transferred to related varieties by traditional plant breeding techniques.
  • the gene is from an alternate plant species than that of said plant, or in some embodiments, the gene is bacterial in origin. In some embodiments, the expression of the gene is inducible.
  • the method further comprises the step of engineering the plant to under-express or to fail to express one or more genes, whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.
  • under-expression or abrogated expression may be effected by any of the many standard means known in the art.
  • reducing expression of a desired gene will utilize expression of antisense transcripts.
  • Antisense RNA has been used to inhibit plant target genes in a tissue-specific manner (e.g., van der Krol et al. Biotechniques 6:958-976 (1988), herein incorporated by reference). Antisense inhibition has been shown using the entire cDNA sequence as well as a partial cDNA sequence (e.g., Sheehy et al. Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); Cannon et al. Plant Mol. Biol.
  • a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed.
  • the expression cassette is then transformed into plants and the antisense strand of RNA is produced.
  • the nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression.
  • the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full-length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred.
  • RNA molecules or ribozymes can also be used to inhibit expression of the target gene or genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • RNAs A number of classes of ribozymes have been identified.
  • One class of ribozymes is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants.
  • the RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch-viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, Solanum nodiflorum mottle virus and subterranean clover mottle virus.
  • the design and use of target RNA-specific ribozymes is described in Haseloff, et al. Nature 334:585-591 (1988).
  • Ribozymes targeted to the mRNA of a lipid biosynthetic gene, resulting in a heritable increase of the target enzyme substrate, have also been described (Merlo A O et al, Plant Cell 10: 1603-1621 (1998), herein incorporated by reference).
  • Another method of reducing expression of a desired gene utilizes the phenomenon of co-suppression or gene silencing (See e.g., U.S. Pat. No. 6,063,947, herein incorporated by reference).
  • the phenomenon of co-suppression has also been used to inhibit plant target genes in a tissue-specific manner.
  • Co-suppression of an endogenous gene using a full-length cDNA sequence as well as a partial cDNA sequence (730 bp of a 1770 bp cDNA) are known (e.g., Napoli et al. Plant Cell 2:279-289 (1990); van der Krol et al. Plant Cell 2:291-299 (1990); Smith et al. Mol. Gen.
  • the introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
  • the introduced sequence in the expression cassette needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that are overexpressers. A higher identity in a shorter than full-length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used.
  • siRNAs can be applied to a plant and taken up by plant cells; alternatively, siRNAs can be expressed in vivo from an expression cassette.
  • RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to target a specific gene product, resulting in post- transcriptional silencing of that gene. This phenomenon was first reported in Caenorhabditis elegans by Guo and Kemphues Cell, 81(4):611-620 (1995) and subsequently Fire et al.
  • RNA interference RNA interference
  • target genes whose under-expression or lack of expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation or a combination thereof.
  • dsRNA used to initiate RNAi may be isolated from native source or produced by known means, e.g., transcribed from DNA.
  • the promoters and vectors described in more detail below are suitable for producing dsRNA.
  • RNA is synthesized either in vivo or in vitro.
  • endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
  • the RNA is provided transcription from a transgene in vivo or an expression construct.
  • the RNA strands are polyadenylated; in other embodiments, the RNA strands are capable of being translated into a polypeptide by a cell's translational apparatus.
  • the RNA is chemically or enzymatically synthesized by manual or automated reactions.
  • the RNA is synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with no or a minimum of purification to avoid losses due to sample processing. In some embodiments, the RNA is dried for storage or dissolved in an aqueous solution. In other embodiments, the solution contains buffers or salts to promote annealing, and/or stabilization of the duplex strands.
  • a cellular RNA polymerase e.g., T3, T7, SP6
  • the RNA may be purified prior to introduction into the cell.
  • RNA can be purified from a mixture by extraction with
  • the dsRNA is transcribed from the vectors as two separate stands.
  • the two strands of DNA used to form the dsRNA may belong to the same or two different duplexes in which they each form with a DNA strand of at least partially complementary sequence.
  • the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different.
  • a DNA duplex provided at each end with a promoter sequence can directly generate RNAs of defined length, and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. No. 5,795,715; incorporated herein by reference. RNA duplex formation may be initiated either inside or outside the cell.
  • Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
  • the length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.
  • the dsRNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more.
  • the dsRNA used in the methods of the present invention is about 1000 bp in length.
  • the dsRNA is about 500 bp in length.
  • the dsRNA is about 22 bp in length.
  • the sequences that mediate RNAi are from about 21 to about 23 nucleotides. That is, the isolated RNAs of the present invention mediate degradation of the target RNA (e.g., major sperm protein, chitin synthase, or RNA polymerase II).
  • the double stranded RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi for the target RNA.
  • the present invention relates to RNA molecules of varying lengths that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi cleavage of the target mRNA.
  • the RNA molecules of the present invention comprise a 3' hydroxyl group.
  • the amount of target RNA is reduced in the cells of the plant exposed to target specific double stranded RNA as compared to cells of the plant or a control plant that have not been exposed to target specific double stranded RNA.
  • knockouts may be generated by homologous recombination. In some embodiments, knockouts may be generated by heterologous recombination. In some embodiments knockouts may be generated by Agrobacterium transfer- DNA. Generally, plant cells are incubated with a strain of Agrobacterium that contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described.
  • T-DNA Agrobacterium transfer-DNA
  • Homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.
  • the invention provides a plant engineered in accordance with the methods of this invention and in some embodiments, the invention provides a seed of a plant or a transgenic plant thus engineered.
  • the plant or seed is of a plant comprising a crop, flowering plant, grain-bearing plant, fruit-bearing plant, nut-bearing plant, herb, turf grass, sod or seedling.
  • such plant or seed is of a plant comprising a banana plant, strawberry plant, blackberry plant, blueberry plant, peach tree, nectarine trees, pear tree, apple tree, grape vine, vegetable plant, pine tree, olive tree, oil palm tree, rubber tree, coffee plant, cotton plant, ornamental plant, flower, flowering, bulb producing plant and others, as will be appreciated by the skilled artisan.
  • the invention relates to methods for promoting boron tolerance in a plant, and for any plant or plant product obtained in accordance with the methods of this invention.
  • Such plants and plant products include, inter alia, crops, fruit, or agricultural product.
  • Crop should be understood to include the plain meaning of the word, and includes cultivated plants and agricultural produce. Crops thus obtained may be cultivated for, e.g., food, medical or industrial use. Crops which may be derived from boron-tolerant plants obtained in accordance with the methods of this invention may include but are not limited to vegetables, e. g., tomatoes, peppers, corn, potatoes, celery; grains, e. g., rice, barley, wheat; nuts, e. g., almonds, pecans, peanuts; and fruit. Other examples of crops which may be derived from boron-tolerant plants obtained in accordance with the methods of this invention include but are not limited to cacao, sugar cane, sugar beets, coffee beans, rubber latex, cotton and flower bulbs.
  • fruits should be understood to include the plain meaning of the word, and includes an edible, usually sweet and fleshy, ovary of a seed-bearing plant or the spore-bearing structure of a plant that does not bear seeds. As such, fruits are a subclass of plant crops or products. Fruits which may be obtained in accordance with the methods of this invention include but are not limited to grapes, bananas, peaches, nectarines, pears, apples, grapefruit, tangerines, lemons, limes, and berries, such as strawberries, blackberries and blueberries.
  • the term "agricultural substance” should be understood to include the plain meaning of the word, and includes plants and their components, such as roots, stems foliage, flowers, etc., crops, harvested crops, and mixtures thereof.
  • Exemplary agricultural substances include vegetable crops and plants, berry fruit crops and plants, berry fruit bushes, flowers, ornamental bushes, pome fruit trees and crops, such as apples and pears, stone fruit trees and crops, such as peaches and plums, grain crops and plants, bulbs, seeds, tubers, turf grass, fruit plants (e. g., bananas), vine crops and plants, tobacco plants, ornamental trees, commodity crops, plants and trees, medicinal plants and herbs.
  • the present invention is not limited to any particular type of plant.
  • said plant is chosen from one or more members of a grass family, a sedge family and a rush family.
  • said plant comprises one or more of annual and perennial plants.
  • the plant is a warm season plant.
  • said warm season plant is a turfgrass.
  • said turfgrass is one or more of bahiagrass, Bermudagrass, centipedegrass, St. Augustine grass, zoysiagrass, carpetgrass, centipedegrass, buffalograss, hurricanegrass, seashore paspalum and the like.
  • the turfgrass of the present invention is not limited to wild-type turfgrass. Indeed a variety of turfgrasses are contemplated.
  • said turfgrass is one or more of a wild- type turfgrass. In some embodiments, said turfgrass is one or more of a sport, selectively bred, and cultivator. In some embodiments, said turfgrass is one or more of a cloned plant, transgenic plant, and the like.
  • the present invention is not limited to any particular type of ornamental grass and ornamental sedge. Indeed a variety of ornamental grasses and ornamental sedges are contemplated. In one embodiment, said ornamental grass is an Indian grass. In one embodiment, said ornamental sedge is one or more of Cyperaceae; for example Carex spp., Scirpus spp., Cyperus spp., and the like.
  • the present invention is not limited to any particular type of rush.
  • said rush is one or more of Juncaceae; for example Juncus spp., Luzula spp., Eleocharis spp., Equisetum spp., Hierochloe spp., Hystrix spp., and the like.
  • the plant is a cold season plant.
  • the present invention is not limited to any particular cold season plant.
  • said cold season plant is a turfgrass.
  • said turfgrass is one or more of bluegrass (e.g. Kentucky bluegrass), tall fescue, Italian ryegrass and perennial ryegrass and the like.
  • said transgenic plant is a fodder plant.
  • said fodder plant is one or more of fescues, Sudan grass, clover, alfalfa, legumes, forage grasses, bentgrass, redtop, fiorin grass (e.g. Agrostis spp.); bluegrass (e.g. Poa spp.); Columbus grass (Sorghum almum); fescue (e.g. Festuca spp.); Napier, elephant grass (Pennisetum purpureum); orchard grass (Dactylis glomerata); Rhodes grass (Chloris gayana); Sudan grass (Sorghum vulgare var. sudanense); Timothy grass (Phleum pratense), and the like.
  • fescues Sudan grass, clover, alfalfa, legumes, forage grasses, bentgrass, redtop, fiorin grass (e.g. Agrostis spp.); bluegrass (e.g. Poa spp.); Columbus grass (Sorghum almum); fescu
  • a legume is one or more of birdsfoot trefoil (Lotus corniculatus); lespedeza (Lespedeza spp.); kudzu (Pueraria lobata); sesbania (Sesbania spp.); sainfoin, esparcette (Onobrychis sativa); sulla (Hedysarum coronarium), and the like.
  • birdsfoot trefoil Lotus corniculatus
  • lespedeza Lespedeza spp.
  • kudzu Pueraria lobata
  • sesbania Sesbania spp.
  • sainfoin esparcette (Onobrychis sativa)
  • sulla Hedysarum coronarium
  • said turfgrass is one or more of a wild-type turfgrass. In some embodiments, said turfgrass is one or more of a sport, selectively bred, and cultivator turfgrass. In some embodiments, said turfgrass is one or more of a cloned plant, transgenic plant, and the like.
  • the present invention is not limited to any particular type of grass, sedge and rush. Indeed a variety of ornamental grass, ornamental sedge and ornamental rush are contemplated.
  • said ornamental grass is an Indian grass.
  • said ornamental sedge is one or more of Cyperaceae; for example Carex spp., Scirpus spp., Cyperus spp., and the like.
  • the present invention is not limited to any particular type of rush.
  • said rush is one or more of Juncaceae; for example Juncuss spp., Luzula spp., Eleocharis spp., Equisetum spp., Hierochloe spp., Hystrix spp., and the like.
  • the present invention is not limited to any particular type vegetative propagation. Indeed a variety of ways to provide vegetative propagation are contemplated.
  • said plant comprises one or more parts for vegetative propagation.
  • said parts for vegetative propagation comprises one or more sprigs, plugs, stolons, rhizomes, callus, meristem and sod.
  • said transgenic plant is a seed. In other embodiments, said transgenic plant is a tiller. In other embodiments said transgenic plant comprises a cold season plant. The present invention is not limited to any particular cold season plant. In one embodiment, said cold season plant is a turfgrass. In some embodiments, said turfgrass is one or more of bluegrass (e.g. Kentucky bluegrass), tall fescue, Italian ryegrass and perennial ryegrass and the like. In other embodiments, said transgenic plant is a fodder plant. In some embodiments, said fodder plant is one or more of fescues, Sudan grass, clover, alfalfa, legumes, forage grasses, bentgrass, redtop, fiorin grass (e.g.
  • Agrostis spp. bluegrass (e.g Poa spp.); Columbus grass (Sorghum almum); fescue (e.g. Festuca spp.); Napier, elephant grass (Pennisetum purpureum); orchard grass (Dactylis glomerata); Rhodes grass (Chloris gayana); Sudan grass (Sorghum vulgare var. sudanense); Timothy grass (e.g. Phleum pratense), and the like.
  • a legume is one or more of birdsfoot trefoil (e.g. Lotus corniculatus); lespedeza (e.g. Lespedeza spp.); kudzu (e.g.
  • Pueraria lobata sesbania (e.g. Sesbania spp.); sainfoin, esparcette (e.g. Onobrychis sativa); sulla (e.g. Hedysarum coronarium), and the like.
  • Thellungiella was demonstrated herein to accumulate much less boron than Arabidopsis, which difference is not due to transpiration; as the transpiration of Thellungiella is not lower than that of Arabidopsis, based both on prior related observations and based on the results provided herein indicating that B-stressed Arabidopsis transpires less than the B-tolerant Thellungiella (Figure 4E).
  • Thellungiella is protected at the shoot level by a cellular mechanism of boron detoxification by complexing boron with, mainly, malic acid and fructose.
  • this invention provides a method for reducing soil boron content, the method comprising:
  • the method further comprises the step of engineering the plant to exhibit altered expression of one or more genes, whose altered expression results in enhanced malic acid, fructose, glucose or citric acid production or accumulation within said shoot or a combination thereof, which in turn forms s intracellular complexes with boron and such an engineered plant is then grown in the soil.
  • the soil is irrigated with desalinated water.
  • the soil bounds or lines a water reservoir.
  • the plants are grown in fields wherein the irrigation water, the soil or the ground water contains higher than desirable levels of boron. Plants are selected to tolerate the boron in the soil or water, and to take up boron while growing in the field.
  • the plant material will incorporate boron into its tissues. Boron may then, inter alia, be removed from the planted area by harvesting and removing plant materials. In some embodiments, the plants may remain at their planted site indefinitely.
  • the harvested plant material must be handled as a waste product and disposed of in a conventional manner.
  • the benefit is the relatively low energy costs of harvesting the plant material compared to recovering leachates or pumping ground water.
  • a second benefit is obtained because the plant materials may be safely used for a variety of finished products.
  • the invention provides, in various embodiments, all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
  • elements are presented as lists, e.g. in Markush group format or the like, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.
  • Plant material Seeds of Arabidopsis thaliana (ecotype Columbia) and Thellungiella halophila (ecotype Shandong) were germinated in a soil mix (from Shaham, Givat Ada, Israel) composed of 30% Finish torf, 30 % vermiculite (size 3), 20% 'tuf (size l-3mm), 20% perlite (size 4). To achieve uniform germination, the seeds in pots underwent cold stratification in the dark under a plastic cover, at 4 °C Arabidopsis for 3 days, Thellungiella for 14 days.
  • the plants germinated practically simultaneously, between two and four days after the end of the cold treatment, and were then grown under a short-day-regime (lOh L / 14 h D) at 350 ⁇ . ⁇ - . ⁇ "1 and at a constant temperature of 21 °C.
  • Tissue sap extraction Fresh plant parts were placed in a syringe cylinder, with several layers of gauze at the bottom. The syringe (without a plunger) was then immersed for a few seconds in liquid N2, then let thaw completely in a 15 mL test tube, and subsequently it was centrifuged at 3,200 g for 5 min. The gauze-filtered liquid sap was collected from the test tube, separately from the pellet which remained in the syringe.
  • Bsmpl is boron concentration in the plant extract sample (in ⁇ g/g, obtained after calibration with standards and subtraction of blank)
  • Vsmpl is the total sample volume (in cm3)
  • Dsmpl is the sample density taken as lg.cm-3)
  • Wpl is the weight (fresh, dry, or fresh-dry, as indicated) of the plant material, in g.
  • [B]int in mmol per kg FW, or per kg "plant water" (FW-DW) Bpl was divided by boron molar weight, 10.81g/mol.
  • Sample preparation 100 of extract was dried under a dry N 2 stream at room temp. The metabolites were treated with silylation reagent (BSA ( ⁇ , ⁇ - bis(trimethylsilyl)acetamide) +TMCS (trimethylchlorosilane) +Pyridine 5: 1 : 15), and the derived material was solubilized with cyclohexan.
  • BSA silylation reagent
  • TMCS trimethylchlorosilane
  • GC-MS analysis The samples were analyzed using Trace GC Ultra gas chromatograph equipped with Thermo TR-5ms SQC (30mx0.25mm, 0.25 ⁇ ) capillary column coupled to Polaris Q ion trap mass selective detector (Thermo Scientific).
  • GC-MS parameters were used in the analysis: carrier gas - helium; gas flow - lml/min; temperature of injector - 270 °C; injection mode - split (1 :50); initial oven temperature was 150 °C (for 1 min) then ramped at 3.57min to 190 °C (held for 4 min), then ramped at 127min to 280°C (held for 5 min); scan range was m/z 50-600.
  • Ethanol extraction 250 mg fresh leaves harvested from Arabidosis and Thellungiella grown in hydroponics and exposed for 7 days to different boron concentrations, were placed in 2 mL test tubes with screw-on caps and 0.5 mL ethanol 80 % was added. The test tubes were heated to 100 °C for 2 minutes, and the extracts were transferred to corresponding new test tubes. The extraction procedure and extract collection were repeated on the plant material twice more, with heating at a lower temperature of 80 °C. The extracts were dried at 40 °C in a chemical hood under a stream of warm air, and 800 mL of filter-sterilized DDW was added to each test tube.
  • test tubes with extract were vortexed, and then centrifuged at about 15,000 g at room temperature.
  • the liquid was decanted and filtered through a 0.45 mm Whatman cellulose acetate syringe filter (CAT#10462100). Subsequently hydrophobic materials were removed from the filtered extract by the addition of 800 mL chloroform, 2 minutes of mixing by vortex, centrifugation (as above) for 10 minutes and decantation of the top water phase with the sugars, without disturbing the lower phase.
  • Sample preparation 50 mL of the extract were dried under a dry N2 stream at 60 °C.
  • the extract was treated with silylation reagent (BSA [N,0-bis(trimethylsilyl)acetamide] +TMCS [trimethylchlorosilane]+Pyridine 5:1 : 15), and the derived material was solubilized with cyclohexan.
  • BSA N,0-bis(trimethylsilyl)acetamide] +TMCS [trimethylchlorosilane]+Pyridine 5:1 : 15
  • a solution of 1 mg B .g-1 FW of diphenyl ether served as a blank standard for polyol quantitation.
  • the hydroponic nutrient solution (based on Smeets et al. 2008, with a small alteration) contained: 0.505 mM KN0 3 , 0.15 mM Ca(N0 3 )*4H 2 0, 0.1 mM NH 4 H 2 P0 4 , 0.1 mM MgS0 4 , 4.63 ⁇ H 3 B0 3 , 2 ⁇ EDFS (CioHi 2 N 2 NaFe0 8 ), 0.91 ⁇ MnCl 2 *4H 2 0, 0.16 ⁇ ZnS0 4 *7H20 , 0.06 ⁇ Na 2 Mo0 4 *2H20, 0.03 ⁇ CuS0 4 , all dissolved in DDW.
  • Figure 1A demonstrates the plantlets growing in rockwool- filled test tubes and Figure IB demonstrates the Root system hanging down from the 4 cm test tubes (lifted out of the aerated nutrient solution for photography and exposing the red air-stones). The roots were gently pried separate while in water, for obtaining the measurements described. Note the relative tolerance of Thellungiella to boron. Similar effects were observed in at least five independent experiments.
  • Thellungiella plants appeared not much different from plants grown in control conditions (Fig. 3).
  • Table 2 Major polyol metabolite concentration in leaf tissue extracts of Arabidopsis (Ara) and Thellungiella (The) exposed to different boron treatments, [B]ext.
  • A Metabolite concentrations (determined using GC-MS, as described) are single determinations. For the recited experiment numbers III-IV listed in Table 2, the metabolite concentrations were determined using GC-MS as described, and expressed as mM in the "plant water", i.e., the difference between the shoot fresh and dry weights, FW-DW. Values presented represent the mean values obtained for 2-3 repeats of each experiment. ND: 'not detected'.
  • the mean RATIOb (the ratio between SUMb and the accumulated [B]int) was significantly higher than '2' only under control conditions (Table 4), suggesting that while Thellungiella abounds in B-binding polyol metabolites relative to Arabidopsis, malic acid and fructose alone would not suffice for neutralizing the accumulated boron completely in 2: 1 polyohB complexes.
  • Lactic_Acid AVG 0.154 0.173 0.359 0.168
  • SUMb is the combined concentration only of malic acid and fructose (M+F), which bind boron with higher stability constants than the other polyols (see text for references).
  • RATIOa and RATIOb correspond to the respective SUMs divided by [B]int determined in each individual experiment. In experiments I and II, all SUM and RATIO values were calculated from single determinations.
  • Ratiob was not similarly consistent in B-treated Thellungiella: although larger than '2' in Expt. I, Ratiob did not exceed '2' in 5 mM in Expts. Ill and IV. In contrast, in B-treated Arabidopsis both RATIOa and Ratiob were always smaller than '2'.
  • RATIOa in Expts. Ill and IV exceeded '2' in Thellungiella and were smaller than '2' in Arabidopsis (a: p ⁇ 0.0005; b: p ⁇ 0.005, c: p ⁇ 0.05; shown also in Fig. 6B).
  • RATIOa of Expts. I and II agreed qualitatively with these results (shown also in Fig. 6C), suggesting together an abundance of polyol metabolites in Thellungiella (but not in Arabidopsis) roughly adequate to bind all accumulated boron at a 2: 1 ratio and thereby alleviate boron toxicity.
  • RATIOb in Expts. I and II was qualitatively consistent with RATIOa.
  • Some of the envisioned methods for developing the columns for use in accordance with the large scale boron removal as herein described include filling columns with crushed filtering material (from either saline-pretreated or from non-pretreated plants), at various packing densities (manipulated by mixing the organic material in different proportions with inert silica sand) at an optimum pH), at various (pump-controlled) flow rates, and determining boron concentration in the column material and in the effluent solution, which can be collected using a fraction collector.
  • the column is then flushed with acidified solution [brackish water acidified with HC1 to pH 5] and the boron concentration in the effluent solution is determined, as well.
  • acidified solution [brackish water acidified with HC1 to pH 5] and the boron concentration in the effluent solution is determined, as well.
  • Some of the methods for developing the columns for use in accordance with the large scale boron removal as herein described include soaking crushed dry leaves of the plants/plant materials as herein described (packed in inert fine-mesh net) in 6 mg/L boron solution (sea-water-like) for various durations, for example, 10, 20 and 60 minutes, on a shaker, at various values of pH: for example, 8, 8.5 both close to that of sea water, and 9.5 (one unit above the pH of sea water, and roughly similar to entry pH in the 2nd stage of sea water desalination), and additionally in 0.6 mg/L boron solution (brackish- water-like), for durations of 20, 60 and 180 minutes at pH between 7.5 (close to the pH of brackish water) and pH 9.5 and determining the values of boron and polyols in the plant material and in the solution using ICP and GCMS, respectively. These experiments may be repeated any number of times to obtain statistically reliable data.
  • Some of methods for developing the columns for use in accordance with the large scale boron removal as herein described include growing Thellungiella for 6-8 weeks for leaf harvest as described in Lamdan et al., 2012 (Plant, Cell & Environment 35:735-746) (and additionally, for about 3 months, to renew seed stocks). In some instances, a dedicated growth chamber is used. Dry weight (DW) is determined after drying in paper bags in an oven for three days at 70 °C. (Fresh weight (FW) of plant leaves will be determined by weighing immediately after their harvest).
  • Thellungiella grown on non-saline water is conducted to assess whether growth in high salinity increases Thellungiella ability to adsorb /to complex B. Saturation plots of material from salt-treated versus non-treated Thellungiella are comparatively assessed.
  • Another large scale boron removal method involves the use of brown algae, such as Saragassum vulgaris, containing a cell-wall-associated polymeric polyol, alginate, known to complex with boron.
  • brown algae such as Saragassum vulgaris
  • a cell-wall-associated polymeric polyol, alginate known to complex with boron.
  • Use of raw dried Saragassum vulgaris algae will be similarly evaluated for the ability to prepare solid supports such as columns for the large scale removal of boron from water sources
  • S. vulgaris is collected, for example, from appropriate sources rocky and the initial boron content (in the dried and crushed algal material) is determined.
  • the dried and crushed algal material is prewashed, to remove the initial boron prior to boron adsorption assays by soaking in (a) mildly- acidified sea water (pH 5 or pH 6, since complexation of boron with polyols decreases with decreasing pH, and (b) mildly-acidified brackish water (also at pH 5 or pH 6; and boron removal is assessed.
  • Sea water or brackish water intended for boron removal is alkalinized to pH 8.5 or 9.5.

Abstract

Cette invention concerne des procédés de réduction de la teneur d'une source d'eau en bore; des supports solides pour réaliser les procédés; des procédés pour augmenter la tolérance au bore d'une plante ou de graines ou de plantes transgéniques produites par les procédés de l'invention. Les procédés de l'invention consistent à créer les conditions grâce auxquelles une pousse de la plante présente des concentrations accrues en acide malique, fructose, glucose, acide citrique ou une combinaison de ceux-ci, ou à créer les conditions grâce auxquelles une pousse de ladite plante contient des complexes intracellulaires d'acide malique, de fructose, de glucose, d'acide citrique ou d'une combinaison de ceux-ci avec du bore, réduisant à son tour la toxicité du bore pour la plante, ce qui en fait un procédé de renforcement de la tolérance de la plante au bore. L'invention concerne en outre des procédés de réduction de la teneur des sols en bore.
PCT/IL2012/050233 2011-07-05 2012-07-04 Matériels végétaux de complexation du bore et leurs utilisations, en référence croisée à des applications connexes WO2013005211A2 (fr)

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CN109174957A (zh) * 2018-09-07 2019-01-11 湖南省农业环境生态研究所 一种重金属镉污染耕地土壤的修复方法
CN109759440A (zh) * 2019-02-27 2019-05-17 安徽科技学院 一种用于修复矿区重金属土壤的绿植种植装置
CN117121923A (zh) * 2023-10-25 2023-11-28 云南五佳生物科技有限公司 一种防治烟草赤星病的组合物、制备方法和应用

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