WO2000066757A1 - Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration - Google Patents

Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration Download PDF

Info

Publication number
WO2000066757A1
WO2000066757A1 PCT/US2000/011400 US0011400W WO0066757A1 WO 2000066757 A1 WO2000066757 A1 WO 2000066757A1 US 0011400 W US0011400 W US 0011400W WO 0066757 A1 WO0066757 A1 WO 0066757A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
freshwater
sample
callus
monocot
Prior art date
Application number
PCT/US2000/011400
Other languages
English (en)
Inventor
Suzanne D. Rogers
Original Assignee
Salem-Teikyo University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Salem-Teikyo University filed Critical Salem-Teikyo University
Priority to CA002370451A priority Critical patent/CA2370451A1/fr
Priority to EP00923627A priority patent/EP1179077A1/fr
Priority to AU43718/00A priority patent/AU4371800A/en
Publication of WO2000066757A1 publication Critical patent/WO2000066757A1/fr

Links

Classifications

    • 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
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • the present invention generally relates generally to the fields of plant molecular biology and bioremediation.
  • Toxic organic compounds can be degraded enzymatically by microorganisms, while toxic metals cannot (Cunningham et al. (1995)). Heavy metals can only be remediated through evacuation to hazardous waste landfills or chemical leaching, remediation technologies that are quite expensive. Even microbial based remediation requires soil extraction. Phytoremediation. using wetland plants to remediate contaminated soils and waters, is a cost effective alternative to traditional cleanup methods.
  • Genes coding for certain remediation activities have been identified (Heaton et al., 1998). Gene transfer methods offer the ability of genetically altering certain plants for targeted remediation activities. For example certain plants have been engineered with a metallothionein gene to accumulate heavy metals.
  • genetically engineered wetland plants have not been used to perform bioremediation processes, probably due to the difficultly of producing genetically engineered wetland plant cells and then producing regenerated plants from these genetically engineered wetland plant cells. Of particular difficulty are methods of transforming these cells, forming callus from these transformed cells, and ultimately regenerating a plant, such as a transgenic plant, from these transformed cells or callus.
  • the present invention provides wetland plants, particularly wetland monocots that can express foreign genes, such as those having bioremediation capabilities, and methods of using them to perform new capacities, such as bioremediation processes for a wide variety of pollutants, including heavy metals, such as mercury.
  • the present invention also provides related benefits as well.
  • FIG. 1A, FIG. IB, FIG. IC, FIG. ID and FIG. IE depict a variety of plasmids and constructs useful in the present invention.
  • FIG. 1A depicts plasmid pBISNl .
  • FIG. IB depicts plasmid pCASl .
  • FIG. IC depicts plasmid pE1120, having ocs-mas "super-promoter *" in pBIlOl . l .
  • FIG. ID depicts plasmid pATC940.
  • FIG. IE depicts plasmid pAL77, which is constructed in pFC3 (see. Lonsdale et al..
  • P.Ubi Maize ubiquitin promoter plus furst intron from pAHC25 (see, Christensen et al., Plant Molecular Biology 18:675-689).
  • FIG. 2A depicts shoot proliferation form J effusus seedling explants on medium supplemented with cytokinins (5 milligrams/1) VA (left). 2iP (middle) and kinetin (right). Media pH were 3.8 (top row) and 7.8 (bottom row). Cultures were grown in standard 60 x 15 mm Petri plates and were photographed after eight weeks in culture media.
  • FIG. 2C depicts rooting of J.
  • FIG. 2D depicts greenhouse established plants made using methods of the present invention, from left to right, Scirpus polyphyllus (one year old), Juncus accuminatus (six months old), Typha angustifolia (eight months old), and Juncus effusus (two years old). Black pots are 23 cm in diameter.
  • FIG. 2E depicts greenhouse established T. latifolia made using methods of the present invention (two years old), in a 30 cm diameter pot.
  • FIG. 3E and FIG. 3F depict the regeneration of T. angustifolia using methods of the present invention.
  • FIG. 3A depicts Typha angustifolia showing callus and somatic embryos after eight weeks in the dark on MS medium with picloram (5 mg/1).
  • FIG. 3B depicts an enlarged view of a somatic embryo.
  • FIG. 3C depicts starch right globular and
  • FIG. 3D depicts bipolar somatic embryos on MS medium with picloram after ten weeks in culture. A blue filter was used in photographic embryos.
  • FIG.3E depicts germinating somatic embryos showing shoots and roots cultured in the light on MS medium with BA (5 mg/1).
  • FIG.3F depicts greenhouse established T. angustifolia plants made using methods of the present invention.
  • FIG. 4A and FIG. 4B depict the regeneration of T. angustifolia using methods of the present invention.
  • FIG.4A depicts germinating somatic embryos of Typha angustifolia cultured in the light on MS medium with GA (5 mg/1) at magnification 2.3X, FIG. 4B at 9.3X.
  • FIG. 5G and FIG. 5H depict the regeneration of T. latifolia using methods of the present invention.
  • FIG. 5A depicts seeds of Typha latifolia (bar is 0.4 cm).
  • FIG. 5B depicts three day old seedling explants used for callus induction (bar is 1 cm).
  • FIG. 5C depicts callus induction from root shoot junction and from root of Typha seedling (bar is 0.15 cm).
  • FIG. 5D depicts nine week old callus growing on MS medium supplemented with 5 mg/1 of BA (bar is 1.5 cm).
  • FIG. 5E depicts shoot regeneration from callus cultured on MS medium supplemented with 5 mg/1 BA (bar is 0.5 cm).
  • FIG. 5F depicts callus showing shoot and root initiation, on medium supplemented with 5 mg/1 (bar is 0.2 cm).
  • FIG. 5G depicts establishment of in vitro regenerated plants in the greenhouse made using methods of the present invention (photograph was taken one month after transfer to pots, bar is 12 cm).
  • FIG. 5H depicts greenhouse established plants made using methods of the present invention showing high root mass (photograph was taken three months after transfer to pots, bar is seven sm).
  • FIG. 6A. FIG. 6B. FIG. 6C, FIG. 6D, FIG. 6E. FIG. 6F, FIG. 6G and FIG. 6H depict the regeneration of T. latifolia and T. angustifolia using methods of the present invention.
  • FIG. 6A depicts three day old seedling explants of T. latifolia used for callus induction at magnification 1.25X.
  • FIG. 6B depicts dark grown nine week old callus of T. latifolia on P5 medium at magnification 1.4X.
  • FIG. 6C depicts shoot regeneration from T. latifolia callus cultured on B5 medium at magnification 2. OX.
  • FIG. 6D depicts eight week old callus of T angustifolia cultured in the light on P5 medium showing somatic embryos at magnification 21.7X.
  • FIG. 6E depicts T. angustifolia callus (top) and somatic embryos (bottom) stained with I2-K where starch rich somatic embryos showing dark blue color, whereas callus did not stain for starch at magnification 20.2X.
  • FIG. 6F depicts five week old calli of T. latifolia (top) and T. angustifolia (bottom) transformed with Agrobacterium containing pBISNl where blue color was developed with a GUS assay, magnification 3X.
  • FIG. 6G and FIG. 6H depict close up views of x-gluc stained calli of T. latifolia (left, magnification 5. OX) and T. angustifolia (right, magnification 13.3X).
  • FIG. 8 depicts regenerated plants, made using methods of the present invention, established in a constructed wetland.
  • A T. angustifolia
  • C J accuminatus
  • D C. lurida
  • E S polyphyllus .
  • FIG. 9A, FIG. 9B. and FIG. 9C depicts regenerated J effusus using methods of the present invention.
  • the present invention recognizes that plant cells, particularly plant cells from freshwater monocot plants, can be transformed and regenerated, particularly to produce plants that have bioremediative capacities.
  • a first aspect of the present invention is a method for transforming a plant cell, preferably a freshwater monocot plant cell such as the freshwater emergent wetland monocots Carex,
  • This aspect of the present invention includes cells and populations of cells, including callus, plants and seeds, made by or derived from this method.
  • a second aspect of the present invention is a method for transforming a plant cell using homologous recombination.
  • the plant cell to be transformed has a nucleic acid sequence that does not naturally occur in the plant cell that can be expressed by the plant cell, such as a reporter gene.
  • a nucleic acid sequence of interest can be targeted to integrate into the locus of such nucleic acid sequence that does not naturally occur in the plant cell using homologous recombination.
  • the nucleic acid sequence of interest can include any gene, but preferably includes a bioremediation gene.
  • a third aspect of the present invention is a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus, Juncus or Typha, preferably Juncus effusus, Juncus accuminatus, Carex lurida or Scirpus polyphyllus :
  • the method includes providing a sample of a plant, inducing shoot development from the sample, and inducing root development from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • a fourth aspect of the present invention is a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus. Juncus or Typha, preferably Typha latifolia or Typha angustifolia.
  • the method includes providing a sample of a plant, forming a callus from the sample, inducing shoot development and inducing root development from the sample. This method can result in the formation of somatic embryos as well as callus.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • a fifth aspect of the present invention is a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus. Juncus or Typha, preferably Juncus accuminatus.
  • the method includes providing a sample of a plant, forming a callus from said sample, inducing shoot development from the sample and inducing root development from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • a sixth aspect of the present invention is a method for regenerating a plant or forming a somatic embryo from a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus, Juncus or Typha, preferably Typha angustifolia.
  • the method includes providing a sample of a plant, forming a callus from said sample, and inducing somatic embryogenesis from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • a seventh aspect of the present invention is a method of bioremediation by exposing a plant made by a method of the present invention to an environment or sample that contains or is suspected of containing at least one contaminant that can be reduced by the plant.
  • isolated polynucleotide' * refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide (1 ) is not associated with the cell in which the isolated polynucleotide is found in nature, or (2) is operably linked to a polynucleotide that it is not linked to in nature.
  • the isolated polynucleotide can optionally be linked to promoters, enhancers, or other regulatory sequences.
  • isolated protein refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which by virtue of its origin the isolated protein (1) is not associated with proteins normally found within nature, or (2) is isolated from the cell in which it normally occurs, or (3) is isolated free of other proteins from the same cellular source, for example, free of cellular proteins), or (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • Polypeptide * ' is used herein as a generic term to refer to a native or non-native protein, fragments thereof, or analogs of a polypeptide sequence.
  • Active fragment refers to a fragment of a parent molecule, such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combinations thereof, that retains at least one activity of the parent molecule.
  • Naturally occurring * refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • Operaably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Control sequences refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes. such control sequences generally include promoter, ribosomal biding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences.
  • the term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Polynucleotide refers to a polymeric form of nucleotides of a least ten bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA or RNA.
  • Genomic polynucleotide refers to a portion of the genome.
  • Active genomic polynucleotide or “active portion of a genome” refer to regions of a gnome that can be up regulated, down regulated or both, either directly or indirecth'. by a biological process.
  • Directly in the context of a biological process or processes, refers to direct causation of a process that does not require intermediate steps, usually caused by one molecule contacting or binding to another molecule (the same type or different type of molecule). For example, molecule
  • A contacts molecule B. which causes molecule B to exert effect X that is part of a biological process.
  • Sequence homology refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, for example 50%, the percentage denotes the proportion of matches of the length of sequences from a desired sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%).
  • “Selectively hybridize” refers to sequences that detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to target nucleic acid strands, under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments thereof and a nucleic acid sequence of interest will be at least 30%, and more typically and preferably of at least 40%. 50%.
  • Hybridization and washing conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, a full length polynucleotide sequence can be labeled and used as a hybridization probe to isolate genomic clones from an appropriate target library as they are known in the art. Typical hybridization conditions and methods for screening plaque lifts and other purposes are known in the art (Benton and Davis. Science 196: 180 (1978); Sambrook et al.. supra, (1989)).
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example. 85% homology means that 85% of the amino acids are 10 identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at least 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff.
  • the term "complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence TATAC corresponds to a reference sequence TATAC and is complementary to a reference sequence GTATA.
  • a reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence can be a subset of a larger sequence, for example, as a segment of a full length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides can each ( 1 ) comprise a sequence (for example a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides. and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a comparison widow refers to a conceptual 1 1 segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence ma) be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window can comprise additions and deletions (for example, gaps) of 20 percent or less as compared to the reference sequence (which would not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window can be conducted by the local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482 (1981)), by the homology alignment algorithm (Needleman and Wunsch, J.
  • the best alignment for example, the result having the highest percentage of homology over the comparison window
  • the best alignment for example, the result having the highest percentage of homology over the comparison window
  • Sequence identity means that two polynucleotide sequences are identical (for example, on a nucleotide-by-nucleotide basis) over the window of comparison. "Percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity .
  • Substantial identity denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more preferably usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25 to 50 nucleotides. wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or additions that total 20 percent or less of the reference sequence over the window of comparison.
  • Substantial identity as applied to polypeptides herein means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, and more preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
  • Constant amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine. alanine, valine. leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutami e; a group of amino acids having aromatic side chains is phenylalanine.
  • tyrosine and tryptophan a group of amino acids having basic side chains is lysine, arginine and histidine; a group of amino acids having acidic side chains is aspartate and glutamate; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine- tyrosine; lysine-arginine; alanine-valine; glutamate-aspartate; and asparagine-glutamine.
  • Label refers to incorporation of a detectable marker, for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • a detectable marker for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • Various methods of labeling polypeptide. nucleic acids, carbohydrates, and other biological or organic molecules are known in the art.
  • Such labels can have a variety of readouts, such as radioactivity, fluorescence, color, chemiluminescence or other readouts known in the art or later developed.
  • the readouts can be based on enzymatic activity, such as that of beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase or luciferase; or radioactive emissions from isotopes such as 3 H, l4 C, 33 S. 12 T or I); fluorescence from fluorescent proteins, such as green fluorescent proteins; or from other fluorescent labels, such as FITC. rhodamine. and lanthanides. Where appropriate, these labels can be the product of the expression of reporter genes, as that term is understood in the art. Examples of reporter genes are beta-lactamase (U.S. Patent No. 5.741.657 to Tsien et al., issued April 21. 1998) and green fluorescent protein (U.S. Patent No. 5.777.079 13 to Tsien et al.. issued July 7, 1998; U.S. Patent No. 5,804.387 to Cormack et al.. issued September 8. 1998).
  • substantially pure refers to an object species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified fraction is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present.
  • object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present.
  • as substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%, 90%, 95% and 99%.
  • the object species or activity is purified to essential homogeneity, (wherein contaminant species or activities cannot be detected by conventional detection methods) wherein the composition consists essentially of a single macromolecular species or activity.
  • an activity may be caused, directly or indirectly, by a single species or a plurality of species within a composition, particularly with extracts.
  • sample means a biological sample, such as a sample derived from a plant.
  • a plant sample can be a whole plant at any stage of development (such as a mature plant, seedling, or the like), a portion of a plant, such as a sample of tissue from at least one portion of at least one plant (such as, for example, leaf, stem or roots), a callus, an immature inflorescence, a seed, an embryo, or a single cell or population of cells, or any combination thereof.
  • Such samples can be obtained using methods known in the art, such as, for example, clipping, culturing. maceration, digestion or other methods.
  • Population of cells means more than one cell.
  • the cells in a population cells can be the same or different, can be a mixture of cells from the same or different organism, and can be a mixture of prokaryotic and eukaryotic cells, such as a mixture of bacteria and plant cells.
  • a population of plant cells can be in the form of dispersed individual cells, in the form of a callus, immature inflorescence, tissue samples from a plant or a portion thereof, samples of embryos or seeds, germinated seedlings, in vitro germinated seedlings, or any combination thereof.
  • “Freshwater wetland monocot plant” means a monocotyledonous plant that can grow in freshwater saturated soils and is preferably a vascular hydrophyte (Mitsch et al., 1993 ). 14
  • “Freshwater emergent wetland monocot plant” means a monocotyledonous plant rooted in soil with part of the plant that emerges from the water, that can grow in freshwater saturated soils and is a vascular phydrophyte (Mitsch et al., 1993).
  • Typha includes all members of that genus.
  • Disarmed Agrobacterium means an Agrobacterium that does not have the activity of forming tumors in plants.
  • Examples of disarmed Agrobacterium, such as Agrobacterium tume actor, include EHA 105 and A281 (Hood et al., 1986) and AT789 and AT793 (Narasimhulu et al.).
  • Vector includes a nucleic acid molecule that is in a configuration (such as a plasmid) and/or packaged (such as in a virus or bacteria) such that at least a portion of the nucleic acid molecule can transfect or transduce a plant cell.
  • a vector can include expression control sequences that are operable in the plant cell.
  • a vector can include a nucleic acid molecule that encodes a gene of interest separate from the expression control sequence.
  • Gene of interest means any nucleic acid molecule that encodes a protein, peptide or polypeptide that has or is believed to have a biological activity, such as bioremediation. therapeutics, or any other biological activity.
  • a gene of interest can be a bioremediation gene and can optionally be operably linked to expression control sequences, wherein such expression control sequences are preferably operable in a plant into which the gene of interest is to be inserted by, for example, transfection or transduction.
  • a gene of interest can also encode an antisense molecule that has or is believed to have a biological activity.
  • Flanking nucleic acid sequences means nucleic acid sequences that are provided on the 3' or 5' end. or both, of a nucleic acid sequence, such as a nucleic acid sequence of interest. Flanking nucleic acid sequences can be attached to a nucleic acid sequence of interest using established methods, such as recombinant methods as they are known in the art.
  • reporter gene means a gene that encodes a polynucleotide or polypeptide that is directly or indirectly detectable.
  • a reporter gene can be a fluorescent protein such as green fluorescent protein that is directly detectable.
  • a reporter gene can be an enzyme whose presence 15 is detected by the conversion of a substrate, such as Xgluc, horseradish peroxidase. beta-lactamase or beta-galactosidase. Appropriate substrates to visualize the presence of such enzymes are readily available.
  • Bioremediation gene is a nucleic acid molecule that encodes a polypeptide or protein that has an activity related to bioremediation.
  • Bioremediation gene nucleic acid sequences from an organisms other than a plant can be modified such that they contain codons preferred by a plant using methods known in the art.
  • Bioremediation genes can also be present on operons.
  • Bioremediation genes include, but are not limited to, genes affecting heavy metal bioremediation relating to contaminants such as copper, mercury, gold, cadmium, lead, telu ⁇ te and silver such as merA, merB, merApe9. merApe20. merApe29, merApe38, merApe47, merT, merP (see. U.S. Patent No. 5,668,294, to Meagher et al., issued September 16. 1997; O'Gara et al.. Appl. Environ.
  • Microbiol. 63:4713-4720 (1997); Chen et al., Appl. Environ. Microbiol. 63:2442-2445 (1997): Jeffrey et al., Microb. Ecol. 32:293-303 (1996); Selifonova et al., Appl. Environ. Microbiol. 60:3503-3507 (1994)); chlorobenzoate, chlorobenzene, toluene and naphthalene such as Tn5271, Tn5542, Tn4653, Tn4651, Tn4656 and Tn4655 (see, Tan, Appl. Microbiol. Biotechnol. 51 : 1-12 (1999)); metallothionein such as HMT1A (see, Kotrba et al., J. Recept. Signal Transduct Res.
  • nitriles such as Nhase (see, Kobayashi et al., Nat. Biotechnol. 16:733-736 (1998)); reduction and oxidation such as cytochromes, such as cytochrome c and P450 (see, Shimoji et al., Biochemistry, 37:8848-8852 (1998); Aubert et al., Appl. Environ. Microbiol. 64: 1308-1312 (1998): Shanker et al., Bioetechnol. Prog. 12:474-479 ( 1996)); 2.4- dichlorophenoxyacetic acid such as tfd (see, Top et al. Antonie Van Leeuwenhoek 73 :87-94
  • Regeneration in the context of regenerating a plant, means regenerating a whole plant, preferably a plant with reproductive capability, from, for example, a portion of a plant, a seeding. an embryo, a callus or a plant cell or a population of plant cells.
  • Plant growth regulator means a chemical or biochemical, such as a polypeptide or small molecule, that acts as a plant growth regulator or regulates plant development and differentiation for the purposes of the present invention.
  • a plant growth regulator can be an auxin, such as, but not limited to, napthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino- 3,5,6-tricholoropicolinic acid (picloram).
  • a plant growth regulator can be a cytokinin, such as. but not limited to, N6-benzyladenic (BA). N6-(2-isopentenyl)-adenine (2iP).
  • l-phenyl-3-(1.2.3- thiodiazol-5-yl)-urea thidiazuron
  • 6-furfurylaminopurine kinetin
  • Other plant growth regulators as they are known in the art or later developed, such as dicamba. are included as plant growth regulators.
  • Inducing shoot development means providing culture conditions, such as nutrients, plant growth regulators, light, temperature or minerals such that a plant sample produces shoot structures.
  • Inducing root development means providing culture conditions, such as nutrients, plant growth regulators, light, temperature or minerals such that a plant sample produces root structures.
  • nducing callus formation means providing culture conditions, such as nutrients, plant growth regulators, light, temperature or minerals such that a plant sample produces a callus. 17
  • the present invention recognizes that plant cells, particularly plant cells from freshwater monocot plants, can be transformed and regenerated, particularly to produce plants that have bioremediative capacities.
  • the present invention includes several general and useful aspects, including:
  • a method for transforming a plant cell preferably a freshwater monocot plant cell such as the freshwater emergent wetland monocots Carex, Scirpus, Juncus or Typha;
  • Scirpus polyphyllus 4) a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus, Juncus or Typha, preferably Typha latifolia; 5) a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex. Scirpus, Juncus or Typha, preferably Juncus accuminatus;
  • the present invention includes a method for transforming a plant cell, preferably a freshwater monocot plant cell such as the freshwater emergent wetland monocots Carex. Scirpus. Juncus or Typha.
  • This aspect of the present invention includes cells and populations of cells, including callus, plants and seeds, made by or derived from this method.
  • the method for making a transformed a plant cell includes providing at least one cell from a plant, and inoculating a plant cell with at least one Agrobacterium. Plants
  • Plants useful in the present invention include freshwater monocot plants, freshwater wetland monocot plants and freshwater emergent wetland monocot plants.
  • the plant is a member of the genus Carex, Scirpus. Juncus or Typha and more preferably is Juncus effusus.
  • the cell used in this method can be any cell from a plant, such as a single cell, a population of cells, an immature influorescence, a sample of callus, a portion of a plant, a seedling a seed or an embryo. Plant cells can be obtained using methods known in the art for a particular type of sample, such as cutting to obtain portions of plants.
  • the Agrobacterium used in the present invention is preferably a disarmed Agrobacterium, such a Agrobacterium tumefaciens, such that i e Agrobacterium does not induce tumor formation in the plant cells.
  • the genes responsible or related to such tumor formation are inactive or removed from the Agrobacterium.
  • the Agrobacterium contains nucleic acid sequences that become integrated in the plant cell ' s genome such that the plant cell becomes stably transfected.
  • nucleic acid sequences from the Agrobacterium can remain extrachromosomal as to the plant cell ' s genome, whereby the plant cell can be transiently or stably transfected.
  • the Agrobacterium used in this method preferably contains a vector, such as those exemplified in the FIG. 1 A through FIG. IE and the Examples.
  • the vector contains a gene of interest and expression control sequences that drive the expression of the gene of interest.
  • Agrobacterium need not be used to introduce 20 a vector into a plant cell, such as when other methods, such as microballistics and lipofection can be used.
  • the gene of interest contains nucleic acid sequences that include codons that are preferred by the plant and the expression control sequences are operative in the plant.
  • the expression control sequences can be chosen such that they drive the expression of the gene of interest in certain tissues, such as root, stems or leaves, or under certain conditions, such as nutrient deprivation.
  • the vector can also include splice donor and/or splice acceptor sites, such that the vector preferably becomes integrated into the plant cell's genome (random integration or directed integration) but that need not be the case (see. WO 98/13353 to Whitney et al., published April 2, 1999; U.S. Patent No. 5.298,429 to Evans et al, issued March 29. 1994); Skarnes et al.. Genes and Development 6:903-918 (1992); Reddy et al., Proc. Natl. Acad. Sci. USA 89:6721 - 6725 ( 1992); Kuspa et al., Proc. Natl. Acad. Sci. USA 89:8803-8807 (1992); Reid et al., Mol.
  • the vector can also contain flanking nucleic acid sequences that can promote the integration of the vector into the plant cell ' s genome via homologous recombination (see, WO 96/30540 to Smith et al., published October 3, 1996).
  • the gene of interest can be any gene, preferably a reporter gene such as, but not limited to, uidA or GUS as in the plasmid vectors depicted in FIG. 1A through FIG. IC or merA (FIG.
  • a bioremediation gene can be fused to a DNA regulatory sequence of interest such as the ocs-mas promoter pATC940 (FIG. ID).
  • the gene of interest can also preferably be a bioremediation gene.
  • the gene of interest does not naturally occur in the plant, but that need not be the case.
  • More than one gene of interest can be provided in a vector.
  • a vector can include a reporter gene operably linked to a bioremediation gene such that both are expressed in the transformed cell. The expression of the reporter gene and/or the bioremediation gene can be driven by expression control sequences on the plasmid or in the plant cell ' s genome.
  • the reporter gene serves the duel purpose or reporting that the vector is operating in the cell, and also reports that the bioremediation gene is also presumptively being expressed in the cell. That the bioremediation gene is being expressed and is active can be determined using methods known in the art. such as Northern blotting for the mRNA derived from the bioremediation gene and assaying for the activity of the protein encoded by the bioremediation gene. 21
  • Agrobacterium instead of using Agrobacterium to deliver a vector to a plant cell, other methods known in the art can also be used. For example microballistics and electroporation can also be used for this purpose. Cells, Callus. Somatic Embryos, Plants and Seeds
  • the present invention includes cells, populations of cells, callus, somatic embryos, plants and seeds made by a method of the present invention.
  • Transformed cells made by the present invention can be cultured to form populations of cells using methods known in the art.
  • the population of cells can be a callus.
  • a population of cells can be of a single clonal population, or a combination of two or more cell types, such as transformed and non-transformed cells.
  • Plants can be regenerated from cells made using the present invention using culturing methods, particularly those taught herein. Mature plants derived from the methods of the present invention can also be used to produce seeds, which are part of the present invention as well.
  • the cells, population of cells, callus, immature inflorescence, somatic embryos, plants and seeds either express the gene of interest or are capable of expressing the gene of interest under a set of conditions. Not all cells of a population of cells, callus, immature inflorescence, somatic embryos, plant or seed need express the gene of interest.
  • tissue expression patterns in a plant can be noted due to different expression control sequences being active in different tissues at different times, particularly in response to environmental factors and conditions.
  • the present invention also includes a method for transforming a plant cell using homologous recombination.
  • the plant cell to be transformed has a nucleic acid sequence that does not naturally occur in the plant cell that can be expressed by the plant cell, such as a reporter gene.
  • a nucleic acid sequence of interest can be targeted to integrate into the locus of such nucleic acid sequence that does not naturally occur in the plant cell using homologous recombination.
  • the nucleic acid sequence of interest can include any gene, but preferably includes a bioremediation gene.
  • a cell of the present invention that expresses a gene of interest can be used as the basis of another aspect of the present invention.
  • Homologous recombination can be used in order to insert another gene of interest, such as a bioremediation gene, at the locus of the reporter gene.
  • a gene of interest is flanked with nucleic acid sequences that target the reporter gene and/or expression control sequences linked thereto, if they are present.
  • This construct can form a vector, which is then used to transform the cell that is expressing the reporter gene.
  • the vector with the gene of interest will. on occasion, insert by homologous recombination at the site of the reporter gene.
  • the inserted gene of interest will insert in-frame, will be expressed, and the expression product will have the desired biological activity.
  • the homologous recombination event will preferably knock-out the activity of the reporter gene.
  • cells that lose reporter gene activity will presumptively have undergone homologous recombination at the site of the reporter gene.
  • This event can be screened for using a variety of methods, including those described herein.
  • the expression of the gene of interest in the cells can be determined by methods known in the art, such as Northern blot analysis to detect mRNA corresponding to the gene of interest and screening for the activity associated with the gene of interest.
  • the reporter gene will not be knocked out, and the cells can be screened for the activity associated with the gene of interest.
  • the vector used for the homologous recombination can include a selectable marker, such as antibiotic resistance. Cells that express the selectable marker would be protected from the antibiotic. Populations of cells that survive exposure to the antibiotic can then be screened for the activity associated with the gene of interest.
  • a cell made by the method of the present invention can be used to make a wide variety of cells that express a variety of genes of interest, such as one or more bioremediation genes. These cells can be cultured to form populations of cells, including callus. Plants can be regenerated from these cells using a variety of methods, including those described herein. Plants and seeds from these regenerated plants can also be obtained. Such cells, population of cells, callus, somatic embryos, immature inflorescence, plants and seeds are part of the present invention. III METHOD FOR REGENERATING A PLANT - A
  • the present invention includes a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus, Juncus or Typha, preferably Juncus effusus. Carex lurida or Scirpus polyphyllus .
  • the method includes provided a sample of a plant, inducing shoot development from the sample, and inducing root development from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • the plant is preferably a freshwater monocot plant, a freshwater wetland monocot plant or a freshwater wetland monocot plant is a freshwater emergent wetland monocot plant.
  • the plant is preferably a member of the genus Carex, Scirpus, Juncus or Typha and is preferably Juncus effusus, Carex lurida or Scirpus polyphyllus.
  • the sample of plant used in this method can be a cell, a population of cells, a portion of a plant, a callus, an embryo, an immature inflorescence, a seedling, an in vitro germinated seedling or any other sample of a plant that includes at least one viable plant cell.
  • the sample preferably is derived from a transgenic plant cell, and is most preferably is derived from a transgenic plant made by a method of the present invention. Inducing Shoot Development
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams L of sucrose.
  • MS Murashige and Skoog inorganic salts medium
  • the media preferably includes between about 0.1 mg/L and about 100 mg/L, more preferably between about 1 mg/L and about 10 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably a cytokinin.
  • the cytokinin is preferably one or more of the following: N ⁇ -benzyladenic (BA).
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured under continuous illumination until 24 shoot development is noted.
  • Shoot development can take between about 10 days and about 8 weeks, preferably between about 2 weeks and about 4 weeks. Inducing Root Development
  • Root development in the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L of sucrose.
  • MS Murashige and Skoog inorganic salts medium
  • the media preferably includes between about 0.01 mg/L and about 50 mg/L, more preferably between about 0.1 mg/L and about 5 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably an auxin.
  • the auxin is preferably one or more of the following: naphthaleanic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), 3.6- dichloro-0-anisic acid (dicamba) and 4-amino-3,5,6-trichloropicolinic acid (picloram).
  • the medium preferably includes one or more of the following. Between about 0.1 % and about 10%), preferably between about 0.5% and about 2% of powdered charcoal. Between about 0.5 milligrams/L and about 500 milligrams/L, preferably between about 5 milligrams/L and about
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous light until root development is noted. Root development can take between about 5 days and about 8 weeks (FIG. 2).
  • the rooted plants are then transferred to cultivation media, such as natural or artificial potting soil in appropriate containers.
  • cultivation media such as natural or artificial potting soil in appropriate containers.
  • the potted plants are cultivated under appropriate conditions of temperature, humidity, light and other factors associated with a particular plant. Mature plants can produce seeds. These plants and seeds are also aspects of the present invention- 25
  • the present invention also includes a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex. Scirpus. Juncus or Typha, preferably Typha latifolia or Typha angustifolia.
  • the method includes providing a sample of a plant, forming a callus from the sample, inducing shoot development and inducing root development from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • the plant is preferably a freshwater monocot plant, a freshwater wetland monocot plant or a freshwater wetland monocot plant is a freshwater emergent wetland monocot plant.
  • the plant is preferably a member of the genus Carex. Scirpus. Juncus or Typha and is preferably Typha latifolia.
  • the sample of plant used in this method can be a cell, a population of cells, a portion of a plant, a callus, an embryo, an immature inflorescence, a seedling, in vitro germinated seedling or any other sample of a plant that includes at least one viable plant cell.
  • the sample preferably is derived from a transgenic plant cell, and is most preferably is derived from a transgenic plant made by a method of the present invention.
  • Callus formation from the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose and vitamins (thiamine (Bl ). nicotinic acid (B3) and pyridoxine (B6)).
  • the media preferably includes between about 0.01 mg/L and about 50 mg/L. more preferably between about 0.1 mg/L and about 5 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably an auxin.
  • the auxin is preferably one or more of the following naphthaleanic acid (NAA), 2.4-dichlorophenoxyacetic acid (2.4-D). 3,6-dichloro-0-anisic acid (dicamba) or 4-amino-3.5.6-trichloropicolinic acid (picloram). 26
  • NAA naphthaleanic acid
  • 2.4-D 2.4-dichlorophenoxyacetic acid
  • dicamba 3,6-dichloro-0-anisic acid
  • picloram 4-amino-3.5.6-trichloropicolinic acid
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous darkness until callus develops, which can take between about 1 week and about 9 weeks (FIG. 3 through FIG. 6).
  • Inducing Shoot Development and Inducing Root Development Shoot development and root development in the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose.
  • the media preferably includes between about 0.1 mg/L and about 100 mg/L, more preferably between about 1 mg/L and about 10 mg/L of at least one plant growth regulator
  • the plant growth regulator is preferably a cytokinin.
  • the cytokinin is preferably one or more of the following: N6-benzyladenic (BA), N6- (2-isopentenyl)-adenine (2iP) and 6-furfurylaminopurine (kinetin).
  • BA N6-benzyladenic
  • INP N6- (2-isopentenyl)-adenine
  • Kinetin 6-furfurylaminopurine
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous darkness until shoot development and root development is noted.
  • Shoot development can take between about 10 days and about 8 weeks.
  • Root development can take between about 5 days and about 8 weeks (FIG. 3). Cultivating. Plants and Seeds
  • the rooted plants are then transferred to cultivation media, such as natural or artificial potting soil in appropriate containers.
  • the potted plants are cultivated under appropriate conditions of temperature, humidity, light and other factors associated with a particular plant. Mature plants can produce seeds. These plants and seeds are also aspects of the present invention.
  • the present invention includes a method for regenerating a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus. Juncus or Typha, preferably Juncus accuminatus .
  • the method includes providing a sample of a plant, forming a callus from said sample, inducing shoot development from the sample and inducing root development from the sample.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom.
  • the plant is preferably a freshwater monocot plant, a freshwater wetland monocot plant or a freshwater wetland monocot plant is a freshwater emergent wetland monocot plant.
  • the plant is preferably a member of the genus Carex, Scirpus, Juncus or Typha and is preferably Juncus accuminatus or Typha angustifolia.
  • the sample of plant used in this method can be a cell, a population of cells, a portion of a plant, a callus, an embryo, an immature inflorescence, a seedling, in vitro germinated seedling or any other sample of a plant that includes at least one viable plant cell.
  • the sample preferably is derived from a transgenic plant cell, and is most preferably is derived from a transgenic plant made by a method of the present invention.
  • Forming a Callus Callus formation from the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose and vitamins (B 1. B3 and B6).
  • the media preferably includes between about 0.01 mg/L and about 50 mg/L, more preferably between about 0.1 mg/L and about 5 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably an auxin.
  • the auxin is preferably one or more of the following: naphthaleanic acid (NAA), 2.4- dichlorophenoxyacetic acid (2.4-D), 3,6-dichloro-0-anisic acid (dicamba) and 4-ammo-3.5.6- trichloropicolinic acid (picloram).
  • NAA naphthaleanic acid
  • 2.4-D 2.4- dichlorophenoxyacetic acid
  • dicamba 3,6-dichloro-0-anisic acid
  • picloram 4-ammo-3.5.6- trichloropicolinic acid
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous darkness until callus develops, which can take between about 1 week and about 4 weeks.
  • the sample can be cultured on an appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose.
  • MS Murashige and Skoog inorganic salts medium
  • the media preferably includes between about 0.1 mg/L and about 100 mg/L. more preferably between about
  • the plant growth regulator is preferably a cytokinin.
  • the cytokinin is preferably one or more of the following: N6-benzyladenic (BA). N6-(2-isopentenyl)-adenine (2iP) and 6- furfurylaminopurine (kinetin).
  • BA N6-benzyladenic
  • 2iP N6-(2-isopentenyl)-adenine
  • kinetin 6- furfurylaminopurine
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous illumination until shoot development is noted. Shoot development can take between about 1 week and about 4 weeks.
  • Root development in the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose.
  • MS Murashige and Skoog inorganic salts medium
  • the media preferably includes between about 0.01 mg/L and about 50 mg/L. more preferably between about 0.1 mg/L and about 5 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably an auxin.
  • the auxin is preferably one or more of the following: naphthaleanic acid (NAA), 2,4-dichlorophenoxyacetic acid (2.4-D). 3.6- dichloro-0-anisic acid (dicamba) and 4-amino-3,5,6-trichlorop ⁇ colinic acid (picloram).
  • NAA naphthaleanic acid
  • dicamba 2,4-dichlorophenoxyacetic acid
  • dicamba 3.6- dichloro-0-anisic acid
  • dicamba 4-amino-3,5,6-trichlorop ⁇ colinic acid
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous illumination until root development is noted. Root development can take between about 5 davs and about 4 weeks. Cultivating, plants and seeds
  • the rooted plants are then transferred to cultivation media, such as natural or artificial potting soil in appropriate containers.
  • cultivation media such as natural or artificial potting soil in appropriate containers.
  • the potted plants are cultivated under appropriate conditions of temperature, humidity, light and other factors associated with a particular plant.
  • Mature plants can produce seeds. These plants and seeds are also aspects of the present invention.
  • the present invention includes a method for regenerating a plant or forming a somatic embryo from a plant, such as a freshwater monocot plant, preferably a Carex, Scirpus. Juncus or Typha, preferably Typha angustifolia.
  • the method includes providing a sample of a plant, forming a callus from said sample and inducing the formation of at least one embryo.
  • This aspect of the present invention includes plants made by this method and seeds derived therefrom. Plants
  • the plant is preferably a freshwater monocot plant, a freshwater wetland monocot plant or a freshwater wetland monocot plant is a freshwater emergent wetland monocot plant.
  • the plant is preferably a member of the genus Carex, Scirpus, Juncus or Typha and is preferably Typha angustifolia.
  • the sample of plant used in this method can be a cell, a population of cells, a portion of a plant, a callus, an embryo, an immature inflorescence, a seedling, in vitro germinated seedling or any other sample of a plant that includes at least one viable plant cell.
  • the sample preferably is derived from a transgenic plant cell, and is most preferably is derived from a transgenic plant made by a method of the present invention. Forming a Callus
  • Callus formation from the sample of the plant is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose and optionally vitamins (Bl . B3 and B6).
  • the media preferably includes between about 0.01 mg/L and about 50 mg/L, more preferably between about 0.1 mg/L and about 5 mg/L of at least one 30 plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably an auxin.
  • the auxin is preferably one or more of the following: naphthaleanic acid (NAA). 2.4-dichlorophenoxyacetic acid (2,4-D), 3-6-dichloro-0-anisic acid (dicamba) and 4- amino-3,5.6-trichloropicolinic acid (picloram).
  • NAA naphthaleanic acid
  • 2,4-D 2.4-dichlorophenoxyacetic acid
  • dicamba 3-6-dichloro-0-anisic acid
  • picloram 4- amino-3,5.6-trichloropicolinic acid
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous darkness or continuous light until callus develops, which can take between about 1 week and about 4 weeks.
  • Inducing Formal ion of Embryos Somatic embryogenesis from the callus is preferably accomplished using culturing methods and medium that utilize plant growth regulators.
  • the sample can be cultured on any appropriate medium, preferably Murashige and Skoog inorganic salts medium (MS) at full strength, supplemented with between about 10 grams/L and about 40 grams/L sucrose.
  • the media preferably includes between about 0.1 mg/L and about 100 mg/L, more preferably between about 1 mg/L and about 10 mg/L of at least one plant growth regulator (if more than one plant growth regulator is used, then the preferred ranges indicate the total concentration of the plant growth regulators).
  • the plant growth regulator is preferably a cytokinin.
  • the cytokinin is preferably one or more of the following: N6-benzyladenic (BA), N6-(2-isopentenyl)-adenine (2iP) and 6- furfurylaminopurine (kinetin). preferably BA.
  • BA N6-benzyladenic
  • 2iP N6-(2-isopentenyl)-adenine
  • kinetin 6- furfurylaminopurine
  • the sample in or on this medium is cultured at an appropriate temperature in the presence or absence of light, or a combination thereof.
  • the samples are preferably cultured in continuous illumination until embryogenesis is noted, which can take between about 1 week and about 4 weeks. Cultivating, plants and seeds
  • the somatic embryos are then transferred to cultivation media for gemination, such as natural or artificial potting soil in appropriate containers.
  • the potted plants are cultivated under appropriate conditions of temperature, humidity, light and other factors associated with a particular plant. Mature plants can produce seeds. These plants and seeds are also aspects of the present invention.
  • the present invention also includes a method of bioremediation by exposing a plant made by a method of the present invention to an environment or sample that contains or is suspected of containing at least one contaminant whose concentration in the environment or sample can be reduced by the plant by, for example, uptake, sequestering, or volatilisation by said plant.
  • a plant can reduce the concentration of at least one contaminant by removing the at least one contaminant by, for example, volatilization, or can concentrate the at least one contaminant within, on or around the plant by, for example, uptake, concentration or sequestering of the at least one contaminant.
  • One aspect of this method of bioremediation includes: providing a plant made by a method of the present invention, and exposing said plant to an environment containing or suspected of containing at least one contaminant that can be uptaken, sequestered, volatilized or reduced by said plant; wherein the amount of said at least one contaminant in said environment is reduced.
  • a second aspect of this method of bioremediation includes: providing a plant made by the method of the present invention; exposing said plant to a sample containing or suspected of containing at least one contaminant that can be uptaken, sequestered, volatilized or reduced by said plant; wherein the amount of said at least one contaminant in said sample is reduced by said plant.
  • plants of the present invention are exposed to an environment such as a local environment (for example, water, sediment, soil or the like) or sample containing or suspected of containing at least one contaminant, whereby the plant can cause a decrease in the amount of the contaminant in the environment.
  • a local environment for example, water, sediment, soil or the like
  • sample containing or suspected of containing at least one contaminant for example, water, sediment, soil or the like
  • the plant and the environment should be chosen so that one suites the other. For example, if the environment is a wetland, then a wetland plant should be chosen.
  • the use of perennial and annuals is a matter of choice to the skilled artisan.
  • the type of climate should be considered. For example, certain plants cannot tolerate harsh winters and may not survive year-to-year. This characteristic may desirable or undesirable, depending on the particular circumstances.
  • the at least one contaminant is in the soil, sediment or water, or any combination thereof.
  • the bioremediation gene in the plants can be in any part of the plant, but preferably localize in the roots of the plant.
  • the plant can accumulate or transform a contaminant. If the plant accumulates a contaminant, then after an appropriate period of time the plant is removed to remove the contaminant for the site or the sample. If the plant transforms the contaminant, such as in the case of mercury mer genes, then the plant need not be removed from the site or sample to remove the contamination from the site or sample.
  • the plant can be placed in a natural environment, or can be placed in an artificial environment.
  • An artificial environment includes an environment modified such that the plant can perform its intended function.
  • An environment can be changed by altering the terrain of the land to improve or modify, for example, water flow or drainage.
  • An artificial environment can also be a separate from the environment, such as in a greenhouse or other structure or location. For example, dredgings or soil from contaminated sites can be moved to an artificial environment or a modified environment where at least one plant of the present invention can be grown such that at least one contaminant therein is reduced.
  • the ability of a plant and method of the present invention to reduce the amount of at least one contaminant in an environment or sample can be determined by measuring the amount of contaminants in an environment or sample over time in the presence and absence of the plant.
  • EXAMPLE 1 SEED ISOLATION AND GERMINATION
  • This example establishes methods for obtaining seeds and initiating germination thereof.
  • Typha lativolia Broadleaf Cattail
  • Typha angustifolia Narrowleaf Cattail
  • seeds were separated from the fuiting spikes by blending with water in a Warring blender. The blended mixture was poured into a water filled glass tank. The seeds settled to the bottom and were collected using a pipette.
  • Juncus accuminatus (Bulrush).
  • Juncus ffususus (Softrush) and Scirpus polyphyllus Many-leaved Bulrush
  • the seeds were separated from the husk by passing through a metal screen with a mesh of one square millimeter.
  • Carex lurida Lid Sedge
  • the seeds were dehusked by crushing the seeds gently in a mortar and pestle for thirty seconds, followed by rubbing the mixture between the hand palms for five seconds.
  • the seeds were separated from the husks by- shaking the mixture on a piece of paper so that the seeds rolled off.
  • Seeds were surface sterilized by stirring in water containing 0.4% Tween-20 for five minutes followed by thirty minutes in a 30% solution of bleach and 0.1% Tween-20. The seeds were rinsed with sterile water and transferred to sterile conical flasks completely filled with liquid MS basal medium containing vitamins and 3% sucrose (Murashige and Skoog, 1962). Seeds were incubated under continuous light to induce germination (30 micromol / m 2 s, 25° C). Some seeds required 45% bleach for thirty-five minutes, optionally with the addition of a five minute immersion in a 0.1 % solution of mercuric chloride for disinfestation.
  • EXAMPLE 2 CULTURE INITIATION, CULTURE REGENERATION, MEDIA, AND GREENHOUSE AND WETLAND ESTABLISHMENT
  • Example 2 This example establishes culture and greenhouse methods to obtain plant materials for genetic manipulation.
  • the seeds obtained in Example 1 were in vitro germinated to seedlings measuring between five and ten millimeters in length. Seedlings reached this size in three days for Typha and six to eight days for the other plants. Seedlings were cultured on gelled callus induction medium, which was basal medium supplemented with one of the auxins NAA, 2.4-D, dicamba or picloram (each at 1 , 2. 5. and 10 milligrams/L). The effect of pH on these seedlings was evaluated by adjusting the medium to various pH levels and observing the effects thereof.
  • J accuminatus explants were cultured for three weeks and Typha for nine weeks in the dark. Embryogenic structures were stained for the presence of starch using an iodine-potassium iodide (L - KI) solution.
  • L - KI iodine-potassium iodide
  • Seedling were cultured directly on gelled basal medium supplemented with BA, 2iP or kinetin (each at 1 , 5, and 10 milligrams/L) and cultured in the light for shoot development. After six weeks on 2iP medium, multiple shoots of about 1 to 1.5 centimeters in length were separated for rooting. C. lurida and S. polyphyllus shoots were rooted in the same rooting medium used for J- accuminatus. Due to shoot browning, J.
  • T. latifolia shoots rooted by nine weeks on shoot induction medium as has been reported in Distichlis (Straub et al. ( 1989)).
  • T. angustifolia callus cultured on picloram. 2.4-D or dicamba in the dark or in the light, proliferated embryos within about 5 to 10 weeks.
  • Twenty percent of cultures on dicamba (2 milligrams/L) produced somatic embryos within two months. The embryos were starch rich, globular and bipolar. Culturing the embryos on B5 medium promoted germination within two to four weeks, whereas Phragmites embryos germinate on medium without growth regulators (Straub et al., 1988).
  • the rooted plants were acclimatized to greenhouse conditions.
  • J effusus callus upon transfer to cytokinin containing medium, in the light can turn black within seventy-two hours. This type of blacking has been reported in Distichlis callus (Straub et 36 al. (1989)). J. effusus could be regenerated, however, by culturing seedling explants directly on cytokinin media in the light. The optimal preferred concentration of cytokinins was about 5 milligrams/L. 2iP caused the highest regeneration frequency and number of shoots per explant. Kinetin caused the poorest response. A range of medium pH (about 3.8 to about 7.8) was tested for effects on shoot proliferation. At all medium pH ' s, shoots were induced (TABLE 2).
  • the shoot proliferation did not differ significantly at media pH values between about 3.8 and about 5.8 in the presence of 2iP and BA. Proliferation was low at pH 7.8 regardless of cytokinin used. Such an effect of pH on direct shoot induction has not been reported for any other wetland species. The ability of some wetland plants to tolerate a wide range of soil pH may reflect this tolerance in vitro (Guntenspergen et al. ( 1989)). The in vitro generated shoots, upon additional subculture to B5 medium, produced higher numbers of shoots, compared to seedling explants. The regenerated shoots were brown at the base and gradually turned dark.
  • Scirpus polyphyllus and Carex lurida were planted in a freshwater constructed wetland habitat in the early spring (FIG. 7, FIG. 8 and FIG. 9).
  • the constructed wetland is located in north central West Virginia (WV). It is an area where freshwater drainage from a natural watershed collected. The area is periodically or intermittently flooded with shallow water. It supports native emergent and submerged herbaceous plants including, but not limited to, Typha and Juncus. Some of the plants like T. latifolia were established from rhizomes dug from a natural wetland and transplanted in the site. Some plants established naturally from wind, water or animal born seed or other propagules.
  • the plants were planted at the average date when the area was frost-free (in WV they were planted May 1 ). Holes 2 to 3 inches larger than the size of the pot each plant was grown in were dug in an area of the wetland containing shallow standing water (at that time of year). The plants were removed from the pots and placed in the water filled holes, with the level of the plant in the pot set level with the soil bottom of the flooded area.
  • J. effusus was also planted in an open grassy terrestrial area in north-central WV. Holes 2 to 3 inches larger than the pot size were dug. and the plants set in the hole and the root ball covered with soil to the same level as was present in the potted plant. The plants were watered weekly, using a garden hose.
  • EXAMPLE 3 ANTIBIOTICS, BACTERIA STRAINS, PLASMIDS, BOMBARDMENT, CULTURE CONDITIONS AND GUS ACTIVITY
  • This example establishes materials and methods for transfection and selection of piant materials obtained using the methods of Example 1 and Example 2.
  • seedlings were cultured in the dark on callus induction medium containing five milligrams/L of picloram (P5 medium), supplemented with an antibiotic or mercuric chloride.
  • kill curves of non- transformed plant materials were performed with hygromycin, kanamycin and mercuric chloride. Such kill curves were performed using hygromycin at 10, 20, 30. 40, 50. 60 and 75 milligrams/L; kanamycin at 25, 50, 75, 100, 150 and 200 milligrams/L; and mercuric chloride at 1.3. 2.7. 4.0, 5.4 and 6.7 milligrams/L. Cerbenicillin and cefotaxime were tested at 500 and 250 milligrams/L, respectively.
  • pBISNl contains GUS whose expression is driven by the "Super Promoter” (a hybrid of mas and ocs) while pKiwil05 contains GUS whose expression is driven by mas and the 35S promoter. Callus was generated from seedlings cultured in the dark on P5 medium following the general procedures described in these Examples.
  • Agrobacterium was grown to 1 OD at 600 nm in LB medium containing 50 milligrams/L kanamycin at 28°C at 200 RPM shaking. Calli were immersed in the bacteria cultured for five to ten minutes, blotted on sterile filter paper, and cocultivated on P5 medium for two days. These calli were transferred to P5 medium containing 250 milligrams/L of cefotaxime. After culturing for one week in the dark, calli were stained for GUS activity generally following established procedures (Jefferson et al. ( 1987) ).
  • the frequency of transient GUS activity was measured by scoring individual calli having one or more GUS positive sites. Relatively low frequencies of GUS expression (between about 1% and about 6% of calli) were obtained in all plants cocultivated with Agrobacterium containing pKiwil05 with the 35S and mas promoter. Young callus (about one month old) had a higher frequency of transient GUS expression than did older cultures. The highest frequency of transient GUS expression obtained was about 40% in embryonic cultures of T.
  • the differences in expression may have been due to the promoter an or the developmental state of the culture (embryogenic) as is seen in corn, although the inventors expressly do not wish to be limited to any proposed mode of action or mechanism (Schlappi and Hohn (1992)).
  • Microprojectile bombardment transformation using promoters such as 35S, Ubi and ACT1 1 showed relatively low expression of GUS. Frequencies of expression with these promoters were relatively low, between about 1% and about 6%, which correlates with the results obtained from the Agrobacterium studies.
  • cefotaxime was found superior to carbenicilhn as was observed in wheat (Mathias et al. (1986)). In this instance, carbenicilhn induced root production. Mercuric chloride, kanamycin and hygromycin could be used to prevent the growth of T. angustifolia, T latifolia and J. accuminatus and thus are potential selective agents for non-transformed cells (TABLE 4) 40

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention reconnaît que des cellules végétales, en particulier des cellules végétales provenant de plantes monocotylédones d'eau douce, peuvent être transformées et régénérées, notamment pour produire des plantes ayant des capacités de biorestauration. Cette invention concerne des procédés servant à transformer et à régénérer une plante, de préférence une cellule de plante monocotylédone d'eau douce, telle que les monocotylédones de marécage énergeantes d'eau douce Carex, Scirpus, Juncus ou Typha. Cette invention concerne des cellules et des populations de cellules, y compris les cals, les plantes et les graines produites par ce procédé ou dérivées de ce procédé. Cette invention se rapporte également à des procédés de biorestauration par exposition d'une plante de cette invention à un milieu ou à un échantillon qui contient ou est soupçonné de contenir au moins un contaminant pouvant être réduit par cette plante.
PCT/US2000/011400 1999-04-29 2000-04-28 Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration WO2000066757A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002370451A CA2370451A1 (fr) 1999-04-29 2000-04-28 Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration
EP00923627A EP1179077A1 (fr) 1999-04-29 2000-04-28 Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration
AU43718/00A AU4371800A (en) 1999-04-29 2000-04-28 Cloned and engineered plants and methods of use for bioremediation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US13151399P 1999-04-29 1999-04-29
US60/131,513 1999-04-29
US17112799P 1999-12-16 1999-12-16
US60/171,127 1999-12-16

Publications (1)

Publication Number Publication Date
WO2000066757A1 true WO2000066757A1 (fr) 2000-11-09

Family

ID=26829550

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/011400 WO2000066757A1 (fr) 1999-04-29 2000-04-28 Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration

Country Status (4)

Country Link
EP (1) EP1179077A1 (fr)
AU (1) AU4371800A (fr)
CA (1) CA2370451A1 (fr)
WO (1) WO2000066757A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070989A2 (fr) * 2000-03-22 2001-09-27 Vlaamse Instelling Voor Technologisch Onderzoek (Vito) Plantes genetiquement modifiees et cellules vegetales comportant des proteines heterologues de complexation et de transport de metaux lourds
WO2002063024A2 (fr) * 2001-02-05 2002-08-15 The University Of South Carolina Research Foundation Culture totipotente continue de monocotyledones selectionnees
WO2002063023A2 (fr) * 2001-02-05 2002-08-15 The University Of South Carolina Research Foundation Culture continue de tissu regenerable totipotent d'arundo donax (canne de provence), plants et tissus totipotents produits a partir de cette culture
CN112079443A (zh) * 2020-09-17 2020-12-15 新乡学院 一种利用湿地克隆植物的克隆性修复重金属镉污染水体的方法

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS ON STN, FRIDBORG ET AL.: "The effect of activated charcoal on tissue cultures adsorption of metabolites inhibiting morphogenesis" *
DATABASE BIOSIS ON STN, JONES ET AL.: "The role of photosynthesis and oxidative reactions in leaf blackening of protea-neriifolia R. Br. leaves" *
DE BLOCK ET AL.: "The cell biology of plant transformation: Current state, problems, prospects and the implications for plant breeding", EUPHYTICA,, vol. 71, 1993, pages 1 - 14, XP002931327 *
PHYSIOLOGIA PLANTARUM,, vol. 43, no. 2, 1978, pages 104 - 106 *
ROGERS ET AL.: "Gus expression in typha latifolia (Cattail) cells transformed with agrobacterium", IN VITRO CELLULAR AND DEVELOPMENTAL BIOLOGY,, vol. 34, ABSTRACT NO. P-1130, no. 3, PART II, March 1998 (1998-03-01), pages 75A, XP002931325 *
ROGERS ET AL.: "Shoot regeneration and plant acclimatization of the wetland monocot cattail (Typha latifolia)", PLANT CELL REPORTS,, vol. 18, 1998, pages 71 - 75, XP002931323 *
SARMA ET AL.: "High frequency plant regeneration from callus cultures of typha angustifolia", IN VITRO CELLULAR AND DEVELOPMENTAL BIOLOGY,, vol. 34, ABSTRACT NO. P-1126, no. 3, PART II, March 1998 (1998-03-01), pages 74A, XP002931322 *
SARMA ET AL.: "Plant regeneration and multiplication of the emergent wetland monocot juncus accuminatus", PLANT CELL REPORTS,, vol. 17, 1998, pages 656 - 660, XP002931324 *
SCI. HORTIC.,, vol. 50, no. 1-2, 1992, pages 137 - 145 *
WALDEN R: "CELL CULTURE, TRANSFORMATION AND GENE TECHNOLOGY", PLANT BIOCHEMISTRY AND MOLECULAR BIOLOGY, XX, XX, 1 January 1999 (1999-01-01), XX, pages 333 - 343, XP002931326 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070989A2 (fr) * 2000-03-22 2001-09-27 Vlaamse Instelling Voor Technologisch Onderzoek (Vito) Plantes genetiquement modifiees et cellules vegetales comportant des proteines heterologues de complexation et de transport de metaux lourds
WO2001070989A3 (fr) * 2000-03-22 2002-04-11 Vito Plantes genetiquement modifiees et cellules vegetales comportant des proteines heterologues de complexation et de transport de metaux lourds
WO2002063024A2 (fr) * 2001-02-05 2002-08-15 The University Of South Carolina Research Foundation Culture totipotente continue de monocotyledones selectionnees
WO2002063023A2 (fr) * 2001-02-05 2002-08-15 The University Of South Carolina Research Foundation Culture continue de tissu regenerable totipotent d'arundo donax (canne de provence), plants et tissus totipotents produits a partir de cette culture
WO2002063024A3 (fr) * 2001-02-05 2003-10-30 Univ South Carolina Res Found Culture totipotente continue de monocotyledones selectionnees
WO2002063023A3 (fr) * 2001-02-05 2003-11-06 Univ South Carolina Res Found Culture continue de tissu regenerable totipotent d'arundo donax (canne de provence), plants et tissus totipotents produits a partir de cette culture
US6821782B2 (en) 2001-02-05 2004-11-23 University Of South Carolina Research Foundation Sustained totipotent culture of selected monocot genera
US7303916B2 (en) 2001-02-05 2007-12-04 University Of South Carolina Sustained totipotent culture of selected monocot genera
CN112079443A (zh) * 2020-09-17 2020-12-15 新乡学院 一种利用湿地克隆植物的克隆性修复重金属镉污染水体的方法

Also Published As

Publication number Publication date
EP1179077A1 (fr) 2002-02-13
AU4371800A (en) 2000-11-17
CA2370451A1 (fr) 2000-11-09

Similar Documents

Publication Publication Date Title
JP6990653B2 (ja) 迅速な植物形質転換のための方法および組成物
Lindsey et al. Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens
DE69929073T2 (de) Verfahren und Zusammensetzungen für die Transformation von Pflanzen
Chhabra et al. Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants
Yu et al. Agrobacterium‐mediated transformation of creeping bentgrass using GFP as a reporter gene
CN108350465A (zh) 改善植物转化的方法和组合物
EP1171618A2 (fr) Procede de transformation de plante
Cho et al. Development of an efficient marker‐free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1
WO2007049816A1 (fr) Plante capable d'accumuler du phosphate inorganique a un taux eleve et utilisation de ladite plante
CN107002030A (zh) 使用土壤杆菌来转化植物细胞或植物组织的方法、转基因植物、转基因细胞或转基因组织、培养基及转化植物细胞或组织的方法的应用
Zhu et al. Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation
Chai et al. Transgenic plants of colonial bentgrass from embryogenic callus via Agrobacterium-mediated transformation
CN107325161B (zh) 一种与耐低氮胁迫和高盐胁迫相关的蛋白及其编码基因与应用
WO2000066757A1 (fr) Plantes clonees et genetiquement modifiees et leur procede d'utilisation pour la biorestauration
Kamo et al. Biolistic-mediated transformation of Lilium longiflorum cv. Nellie White
EP2150100B1 (fr) Procédé de micropropagation de monocotylédones basé sur des cultures cellulaires totipotentes entretenues
DE69932082T2 (de) Verfahren zur verbesserung der effizienz agrobakterium-vermittelter transformation von pflanzen
JP2007312635A (ja) クエン酸分泌能が強化された樹木およびその作出方法
CN102108362B (zh) 一种优化的三苯甲烷还原酶基因及其表达和应用
US8507758B2 (en) Markerless transformation
DE10115761A1 (de) Verfahren zur raschen Herstellung von transgenen, einkeimblättrigen Pflanzen
Koga et al. The transformation system in foxglove (Digitalis purpurea L.) using Agrobacterium rhizogenes and traits of the regenerants
Zhao et al. Transformation of modified cowpea trypsin inhibitor gene and anti-bacterial peptide gene in Brassica pekinensis protoplasts mediated by Agrobacterium tumefaciens
CN108192918A (zh) 转伴矿景天SpHMA3基因的重金属超富集转基因工程油菜的创制及应用
JP2009523021A (ja) 陽性選択に基づくヒマワリおよび脂肪種子の新規で効果的な形質転換法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2370451

Country of ref document: CA

Ref country code: CA

Ref document number: 2370451

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 10069098

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 515463

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 2000923627

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000923627

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000923627

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP