WO1991018094A1 - Transformation d'especes vegetales ligneuses au moyen de particules - Google Patents

Transformation d'especes vegetales ligneuses au moyen de particules Download PDF

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Publication number
WO1991018094A1
WO1991018094A1 PCT/US1991/003629 US9103629W WO9118094A1 WO 1991018094 A1 WO1991018094 A1 WO 1991018094A1 US 9103629 W US9103629 W US 9103629W WO 9118094 A1 WO9118094 A1 WO 9118094A1
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Prior art keywords
cells
plant species
tissues
carrier particles
nodules
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PCT/US1991/003629
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English (en)
Inventor
Dennis E. Mccabe
Brent H. Mccown
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Agracetus, Inc.
Wisconsin Alumni Research Foundation
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Publication of WO1991018094A1 publication Critical patent/WO1991018094A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • 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
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the genetic engineering of plants in general and relates- in particular, to a methodology using particle-mediated transformation techniques to achieve the germ line genetic transformation of woody species of perennial plants such as trees.
  • Tissue culture refers to an in vitro culture of living plant cells.
  • Agrobacterium tumefaciens natively harbors a plasmid, referred to as the Ti (tumor-inducing) plasmid, which has the inherent ability to transfer a portion of its DNA (T-DNA) into a target plant cell.
  • Ti plasmids of Agrobacterium tumefaciens By suitable manipulation of the Ti plasmids of Agrobacterium tumefaciens, it is possible to insert a foreign genetic construction into the T-DNA of the Ti plasmid, which is then transformed into susceptible plant cells in tissue culture by the bacterium.
  • Other techniques for transforming individual cells or cells in tissue culture include direct DNA injection and electroporation of plant protoplast cells.
  • One gene which has been inserted into herbaceous plants is a chi eric genetic construction coding for the expression of an insect-toxic portion of the delta-endotoxin protein produced by the soil dwelling microorganism Bacillus thuringiensis, or B.t.
  • Bacillus thuringiensis Bacillus thuringiensis
  • This toxin has been previously found to be specifically toxic only to Lepidopteran insects, i.e., the larvae of moths and butterflies. Since caterpillars are a primary consumer of leaves of tree species grown for fiber, lumber, or energy, the creation of trees having resistance to attack by Lepidopteran larvae by virtue of the insertion of a B.t. gene would be of significant value.
  • the present invention is summarized in that the genetic engineering of woody species of plants involves the use of a particle-mediated transformation technique in which tissues of the woody plants are transformed with DNA carried on small carrier particles.
  • the present invention is also summarized in that poplar trees are created which express in their leaves an insect-toxic amount of the B.t. Lepidopteran specific toxin.
  • Fig. 1 is an illustration of a particle acceleration apparatus useful within the procedure of the present invention.
  • Fig. 2 is an illustration and a restriction map of the plasmid pTVBTGUS.
  • Fig. 3 is a graphical representation of the results of whole plant assays using the transgenic plants of the present invention.
  • Fig. 4 is a graphical representation of the survival of larvae feeding on parts of the transgenic plants created in accordance with the process of the present invention.
  • Fief. 5 is a graphical representation of a whole leaf assay utilizing the transgenic plants of the present invention.
  • Nodules are phenomenon of the tissue culture of plant cells. -Nodules are independent, spherical, dense cells clusters, formed in an in vitro culture, which display a degree of tissue differentiation which distinguish the cultures from callus and other forms of less differentiated cultures which have been created by dedifferentiated cells. Nodule formation is particularly characteristic of woody species such as poplar, but have also been noted as occurring with other herbaceous plant species such as carrots, day lily, and other woody plants such as citrus, spruce, and pine.
  • nodules in poplar A complete description of the formation of nodules in poplar, and the characterization of such nodules, is contained in the text of "Nodule Culture: A Developmental Pathway With High Potential For Regeneration, Automated Micropropagation, and Plant Metabolite Production From Woody Plants," McCown et al. in Genetics Manipulation of Woody Plants, Ed. by Hanover et al., pg. 149-166 (Plenum Publishing 1988), the disclosure of which is hereby incorporated by reference.
  • nodules are particularly attractive for use in this technique since they may be created, manipulated, and selected in relatively large numbers.
  • exogenous, typically chimeric, genetic constructions into the germ line of woody plant species.
  • exogenous genetic construction is preferably DNA from one or more other organisms, whether of the same or different species, which is introduced into the transformed woody plant through.
  • the method of transformation described herein is the use of particle-mediated transformation in which small carrier particles carry the genetic construction into the cells of the plants.
  • the exogenous genetic construction would normally include a coding sequence which codes for the production in the cells of the plant of a transcription product or a protein of interest.
  • the exogenous genetic construction therefor typically includes flanking regulatory sequences effective to cause the expression of the protein, or the transcription product coded by the coding sequence, in the transformed cells of the woody plant species.
  • flanking regulatory sequences are a promoter sequence sufficient to initiate transcription, and a terminator or polyadenylation sequence sufficient to terminate the gene product, whether by termination of transcription or translation.
  • translational enhancers located between the promoter and the coding sequence to assist in the efficiency of the expression of the genetic product, especially the expression of protein products, in the transformed woody plant cells. It is specifically envisioned that gene products other than proteins may also be expressed by the inserted exogenous genetic construction.
  • the inserted construction can express a negative RNA strand, also referred to as an anti-sense strand, effective either to suppress the expression of an endogenous gene in the woody plant species or to inhibit a disease process by a pathogenic organism. It has been specifically found that the creation of chimeric exogenous genetic constructions, and their insertion into transgenic plants, results in traits which are inheritable by the normal sexual reproduction of plants thereafter in a conventional Mendellian fashion.
  • the process of the present invention is intended to make particular use of a procedured apparatus utilizing an adjustable electric discharge device to physically accelerate ' DNA coated onto small particles into plant cells.
  • An apparatus suitable for use within the present invention is illustrated in Figs. 1 and 2.
  • the apparatus consists of a spark discharge chamber 12 into which are inserted two electrodes 14 which are spaced apart by a distance of approximately 1 to 2 millimeters.
  • the spark discharge chamber 12 is a horizontally extending rectangle having two openings 16 and 18 extending out its upward end.
  • the opening 16 is covered by an access plate 20.
  • the opening 18, located on the side of the rectangle of the spark discharge chamber 12 opposite from the electrodes 14, is intended to be covered by a carrier sheet 22.
  • the electrodes 14 are connected to a suitable adjustable source of electric discharge voltage.
  • Such a source of electric discharge voltage would preferably include suitable electric switching connected to a capacitor of the one to two microfarad size range, with the amount of the voltage of the charge introduced onto the capacitor being adjustable, such as through the use of an autotransformer, through a range of perhaps 1 to 50,000 volts.
  • suitable high voltage electric switching (not shown) is provided so that the capacitor can safely be discharged through the electrodes 14 so that the apparatus can be used conveniently by a user.
  • the carrier sheet 22 intended to be placed upon the opening 18 in the spark discharge chamber 12 is a planar sheet of relatively stiff material such as a sheet of aluminized saran coated mylar.
  • a retaining screen 24 Above the opening 18 in the discharge chamber 12, positioned approximately 5 to 10 millimeters above it, is a retaining screen 24.
  • a target surface 26 Placed above the retaining screen 24 at a distance of approximately 5 to 25 millimeters above the retaining screen, is a target surface 26.
  • the target surface 26 can be any suitable culture surface onto which the material to be transformed may readily be placed such as, mostTconveniently, an overturned petri dish into which the plant tissues have been positioned for culture.
  • Copies of the exogenous foreign genetic construction intended to be transformed into the plant tissues is prepared by suitable DNA preparation techniques well known to those of ordinary skill in the art and multiple copies of the genetic construction are made.
  • the copies of the foreign genetic construction, in aqueous solution are then coated onto small particles of a durable dense carrier material, such as gold, the carrier particles typically being in a size range of 1 to 3 microns.
  • the carrier particles with the exogenous genetic construction coated thereon are then placed upon the carrier sheet 22 which is inserted at the proper opening on the top of the spark discharge chamber 12.
  • the target surface 26 including the living plant material thereon is then placed in position above the retaining screen 24.
  • a small droplet of water, preferably 2 to 4 microliters in size, is then placed bridging between the ends of the electrodes 14.
  • the access cover 20 is placed in position on top of the spark discharge chamber 12. At this point the entire apparatus is enclosed in a vacuum chamber and a vacuum is drawn until it is in the range of approximately 500 millimeters of mercury. A supply of helium is continuously bled into the vacuum chamber to replace the atmosphere in the space between the carrier sheet and the target with helium to take advantage of the lower relative density of helium.
  • the initiation of a spark discharge may be initiated by the user between the electrodes 14. This is done by means of the appropriate electric switching which applies the voltage stored in the capacitor across the terminals of the electrodes 14. The force of this electric discharge bridges the spark discharge gap between the electrodes 14 instantly vaporizing the small droplet of water previously placed therebetween. The force of the vaporization of that water creates a Shockwave within the spark discharge chamber 12 which radiates outward in all directions. The impact of the radiating Shockwave upon the carrier sheet 22 propels the carrier sheet 22 upward with great velocity. The upwardly traveling carrier sheet 22 accelerates until it contacts the retaining screen 24.
  • the use of the helium within the vacuum containment for the apparatus provides less drag on the flight of the carrier sheet 22 as well as less force necessary for the Shockwave to propagate the carrier particles to the target tissues.
  • the carrier sheet 22 is retained, and the carrier particles coated with the exogenous genetic construction previously coated thereon fly off of the carrier sheet and travel freely onward toward the target tissues.
  • the small carrier particles then proceed into the cells of the target tissues placed on the target surface 26 and pass freely into the cytosol of the cells placed thereon.
  • the actual momentum of the carrier particles as they impact the surface of the target tissues is adjustable, based upon the voltage of the initial electric discharge applied to the electrodes 14.
  • the velocity by which the particles impact the target can be adjusted, and thus the depth of penetration of the carrier particles into the tissue of the target tissues can be adjusted continuously throughout the range of adjustment provided for the electric voltage applied across the electrodes 14.
  • the apparatus of Figs. 1 and 2 has previously been demonstrated to be useful for the transformation of tissues of various herbaceous plants. It has been found through the work disclosed herein that this apparatus and the procedure for using it may also be appropriately applied to the transformation of woody plant species and can result in-the germline transformation of complete whole, intact and sexually mature tree species. Therefore, the target tissues to be placed upon the target surface 18 in the apparatus of Fig. 1 for use within the present invention would preferably include a regenerable tissue sample of the woody plant species to be transformed. It has been found that a particularly advantageous tissue type for each transformation is a nodule culture of the woody plant species. Other usable tissue types include stem segments, protoplasts, and suspension cell cultures.
  • the particular tissue types placed upon the target surface 28 be one which can be regenerated either readily, or at least with a sufficient frequency, so that whole, intact plants can be recovered through the use of this technique.
  • Such cultures exist for a variety of woody species, such as the model species poplar disclosed and discussed herein.
  • the technique of particle-mediated genetic transformation can be adapted to a wide variety of tissue types in woody species.
  • the use of a tunable electric spark discharge for the motive force in the particle acceleration has proven also to be of great advantage, since the acceleration imparted to the particles can be readily and easily tuned using this technique, thereby facilitating adaptation of the technique to different tissue types.
  • the present technique can be used either with or without a selectable marker.
  • the selectable marker of kanamycin resistance has been used since the resistance gene provides good selection in poplar, and thus adds to the efficiency of the procedure.
  • selection step could have been omitted, since a readily detectable marker gene, beta-glucuronidase was included in the transformation vector.
  • the omission of selection would require the regeneration of more tissues, since the tissues would have to be screened for the presence of the marker, but the transformation of the germ line of the woody plant species could still be achieved.
  • Other selection agents and marker genes are also known to those of skill in the art. -12-
  • the exogenous genetic construction utilized in the examples described herein was the plasmid pTVBTGUS.
  • An illustration of the coding sequences and a restriction map of the plasmid pTVBTGUS is illustrated on Fig. 3.
  • the plasmid pTVBTGUS includes expression cassettes for three separate coding sequences found to be effectively expressible in the cells of plant tissues.
  • One expressible genetic construction is for the gene aminoglycoside phosphotransferase II (neomycin phosphotransferase) gene (NPT II or APH II) commonly used as a selectible marker for kanamycin resistance in plant transformation procedures particularly those utilizing Agrobacterium-mediated transformation techniques.
  • the plasmid pTVBTGUS also includes a gene coding for the expression of the beta-glucuronidase gene (GUS) , which codes for the expression of an enzyme which can be readily detectible by a colorimetric assay as described by Jefferson, Embo J. , 6:3901-3907 (1987).
  • GUS beta-glucuronidase gene
  • pTVBTGUS Also in the plasmid pTVBTGUS is a plant expression vector cassette coding for the expression of B.t. , i.e. the delta endotoxin from Bacillus thuringiensis.
  • the toxins produced by the soil-dwelling microorganism Bacillus thuringiensis have long been recognized to have potential value as selective biological insecticides due to their unique and specific toxicity only to Lepidopteran insects.
  • the entire DNA sequence of this gene, including flanking regions and inferred amino acid sequence ⁇ have been previously published, as have various physical characteristics and features of the expression in its natural host. It has also been reported that deletion analysis of the full length protoxin coded for by the native gene sequence reveal that the amino terminal portion of the gene is sufficient for toxicity. As described in published PCT patent application WO 89/04868, an amino-terminal - ⁇ 180
  • B.t. delta endotoxin gene can be constructed which codes for the expression of a truncated toxin protein which retains the toxicity of the protoxin.
  • the truncated toxin has been found to be effectively expressed in plant cells to confer those cells with toxicity upon ingestion to Lepidopteran insects.
  • the B.t. toxin coding region in pTVBTGUS codes for the same amino acid as the corresponding B.t. coding region found in plasmid pAMVBTS, described in the above-identified PCT patent application WO 89/04868, but the actual nucleotide sequence is different.
  • the first 141 codons have been altered to ⁇ odons which conform to plant codon usage frequencies. This was accomplished by creating overlapping synthetic oligonucleotides for the desired sequence, and then linking the oligonucleotides together to form the first 141 codons of the coding sequence, which was then inserted into the plasmid pAMVBTS.
  • the plasmid pTV4 incorporated into the expression plasmid pTVBTGUS described herein contains the left and right border sequences from Agrobacterium tumefaciens which are necessary to effect plants for transformation using an Agrobacterium-mediated transformation technique. It is believed that those left and right border sequences are unnecessary in an expression cassette utilized within the method of the present invention. However, since this plasmid was constructed for other Agrobacterium-based transformation experiments with other plants, this expression plasmid was conveniently available for use within the practice of the present invention as described herein.
  • the plasmid pTVBTGUS thus has, in addition to antibiotic resistance markers (Amp r and Su r ) three plant expression cassettes. These expression cassettes are for kanamycin resistance (NPT II) , beta-glucuronidase gene (GUS) and for the B.t. toxin. The inclusion of both the NPT II and GUS genes would be redundant for most applications, since either selection or screening of transformant tissues would normally be performed to identify transgenic tissues, but not both.
  • the plasmid pTVBTGUS is a large one, of approximately 18,687 base pairs, and the ability of the procedure described here to insert such a large DNA insert into transgenic plants is indicative of the ability to achieve the same result with other large constructions.
  • Tissues of NC5339 were used in the protoplast, nodule, and stem transformations discussed below, while NC5331 was also used in the nodule transformations.
  • Cells were cultivated in suspension cultures taken from uniform and rapidly growing cultures at the pre-log and early-log phase of growth which was typically three to seven days after subculture.
  • Protoplasts and suspension cultures were plated at a density of 25,000 to 60,000 cells per milliliter which was deemed to be a population high enough to give adequate viable population after blasting and losses during later development and a low enough density to permit rapid cell division and cell colony singulation.
  • Floating discs in the manner described in Russell et al., supra were created with attached cells derived from protoplasts or overlaid suspension cells derived from early log-phase cultures (3 days old) growing in liquid MS medium (Murashige and Skoog, Physiol. Plant, 15:473-497) supplemented with lmg/1 2-4,D and 0.05 mg/1 BA.
  • the discs were placed on five centimeter petri dishes with one disc plated per plate.
  • the plating medium consisted of 10 milliliters of the basic WPM medium (Lloyd and McCown, Proc. Intl. Plant Prop.
  • Each set of plated protoplasts or suspension culture cells were subject to a single transformation event at a voltage of six kilovolts for the spark discharge.
  • the carrier particles utilized were 1 to 3 micron gold particles.
  • the loading of the particles onto the carrier sheet was in a range of 0.025 to 0.05 milligrams per square centimeter.
  • the density of the copies of the foreign genetic construction DNA applied to the carrier particles themselves was approximately 0.1 micrograms of DNA of pTVBTGUS per milligram of gold beads.
  • the process for coating the DNA onto the carrier particles was to suspend 1 microgram DNA in 2 microliters 0.2M EDTA and 10 icroliters of Tris, pH8. 10 milligrams of gold beads were also introduced into the suspension.
  • the suspension was vortexed and dried under a nitrogen stream. Then the dried particles were resuspended in ethanol, coated onto the carrier sheet, and air dried.
  • the cultures were moved to a liquid growth medium in a multi-well plate in the case of protoplasts or were kept on the target plates in the case of suspension cells. All tissues were kept in the dark for 48 hours after blasting.
  • the protoplast cells were cultured on this medium with continual medium refreshment and osmoticum reduction if needed every 7 to 10 days. As the medium was refreshed and antibiotic selection regimen was included at a rate of 20 milligrams per liter of kanamycin added to the standard WPM medium.
  • the dish was moved to a solidified medium consisting of WPM medium supplemented with 0.1 micromolar BA (benzyladenine) , 0.1 micromolar NAA (naphthalene acetic acid) 0.1 micromolar THI (thidiazuron) and 50 milligrams per liter kanamycin.
  • WPM medium WPM medium supplemented with 0.1 micromolar BA (benzyladenine) , 0.1 micromolar NAA (naphthalene acetic acid) 0.1 micromolar THI (thidiazuron) and 50 milligrams per liter kanamycin.
  • Pieces were taken from actively-growing shoot cultures after four to six weeks of growth in in vitro culture.
  • the pieces to be subject to a transformation event were pre " -treated in multiwell plates with three to five pieces per plate by a liquid medium supplemented with 0.1 micromolar BA, 0.1 micromolar NAA and 0.1 micromolar THI.
  • the treated pieces were shaken on a rotary shaker so that the pieces were moving in the inoculating liquid pre-treatment cultures.
  • the pieces were oriented so that the heaviest concentration of " eristematic zones" of each individual culture piece were exposed to the transformation particle path.
  • the plating medium consisted of 10 milliliters of solidified medium as described in conjunction with the transformation of the protoplasts and suspension cell cultures above. Again the tissues were blotted immediately before the transformation event.
  • the tissues were kept and cultivated in the dark for forty-eight hours after the last transformation event. After that the cultures were plated in light and when growth was visible, the plates were flooded with 50 milligrams per liter kanamycin. Some of the cultures were returned to a liquid medium in multi-well plates under 20 to 50 milligrams per liter kanamycin selection.
  • Nodules were cultivated in accordance with the procedures described by McCown et al., supra. The most successful nodules utilized were those created from stabilized shoot cultures and the nodule cultures created in this fashion could be serially and continuously self-replicated to make continuing in vitro cultures of nodules. Nodules were also created from nodular growth of other tissues including stems and callus cultures, and microcalli derived from protoplasts.
  • nodules were from established cultures, prior to the particle-mediated transformation event, the nodules were pre-treated with the cytokinin THI. This was done after preliminary results indicated a lower level of transformation for nodules harvested directly from a high auxin/low cytokinin medium. The remaining nodule cultures were treated from two to six weeks with 0.1 micromolar THI and basic 0.1 micromolar BA and 0.1 micromolar NAA medium. Nodules which were larger than 1.0 millimeters in diameter were harvested, cut in half, and grouped together in one square centimeter target areas in the middle of petri plates in the same fashion as were the internodes.
  • the cut surfaces of the nodules were upwardly exposed when they were plated into the petri plate so as to be exposed to the transforming particle stream.
  • the medium onto which the cut nodules were plated is solidified pre-treatment medium. Again the surface of the nodules was surface blotted with blotting paper prior to the transformation event for drying.
  • the nodules were subjected to a particle-mediated transformation event between one and ten replicates.
  • the timing ' between replicates was six to twelve hours.
  • the apparatus was used with an electric discharge voltage of fourteen kilovolt applied between the electrodes.
  • the carrier particles used were amorphous crystilline gold powder (Englehard) , and consisted of one to five micron particles.
  • the DNA of pTVBTGUS was loaded on the carrier particles at a rate of 0.1 microgram DNA per milligram gold and the coated gold particles were applied to the carrier sheet at a loading of .025 mg per square centimeter.
  • the plated nodules were kept in the dark for forty-eight hours after the last transformation event.
  • the nodules were kept on plates and were subsequently flooded with fifty milligrams per liter kanamycin when initial growth from the cut surface was observed, typically in a time period of seven to ten days. The kanamycin was refreshed as needed.
  • the new green nodules and calli surviving the kanamycin selection were removed from the surfaces of parent nodules when two to three millimeters in diameter and were cultured separately in the same solidified medium.
  • the nodules were cultivated through to further differentiation including shoot and root development into plantlets.
  • the regeneration of such nodules is through organogenesis. Nodule shoot differentiation was stimulated by THI administered as a pulse treatment at a rate of 0.1 micromolar.
  • Adventitious rooting followed resulting in plantlets.
  • the resulting plantlets were cultivated into trees which appeared morphologically normal exhibiting the normal growth characteristics of poplar trees regenerated from nodule culture.
  • regenerate plants were positive for kanamycin resistance, but not for the B.t. toxin gene, as determined by PCR analysis. However, some kanamycin resistant plants assayed positive PCR for both the 5' and 3* ends of the B.t. gene.
  • FIGS 3 - 4 Shown in Figures 3 - 4 are graphical representations of the results of the insect bioassays.
  • a control plant consisting of a regenerated poplar tree was compared to transgenic poplar plants designated BT-1 and BT-2.
  • the whole plants were assayed.
  • Live larvae of forest tent caterpillar (Malacosoma disstria) were placed onto the whole plants to feed upon the leaves.
  • the larvae were placed at a density of six larvae per plant.
  • the larvae were given no other choice for feeding activity and the number of larvae surviving at the end of six days and the mean weight of the larvae was measured for the three plants in the assay.
  • Fig. 3 Shown Fig. 3, Fig. 3, a control plant consisting of a regenerated poplar tree was compared to transgenic poplar plants designated BT-1 and BT-2. The whole plants were assayed. Live larvae of forest tent caterpillar (Malacosoma disstria)
  • Fig. 4 Shown in Fig. 4 is a graphical representation of the survival of larvae from the whole plant assay, a whole leaf assay, and the leaf disk assay.
  • the whole plant assay represented the same replicate as indicated in Fig. 3.
  • the whole leaf assay was an assay conducted in a petri plate with four larvae per dish, no feeding choice, and a feeding time of four days.
  • the leaf disk assay was conducted with two larvae per dish with a choice of disk for the larvae to feed upon, and with a time period of thirty hours.
  • there seem to be little difference in the survival of larvae in a short term trial such as that conducted on the leaf disk while in both the whole plant and whole leaf assays significant differences in survival rates for the transgenic plants as compared to the control plants were evident.
  • Fig. 5 Illustrated in Fig. 5 is a graphical representation of the whole leaf assay in more detail.
  • the left-hand bar graph of Fig. 5 illustrates the mean weight of the four larvae per dish after the four days of treating on the whole leaf from the control or the transgenic plants.
  • the right-hand bar graph in Fig. 5 illustrates the area eaten in square centimeters of the leaf in the assay by the feeding larvae.
  • the amount of the leaf eaten was a very small fraction of the amount of the leaf tissue eaten from the control plant.
  • the larvae were not killed since they had an avoidance behavior to eating the leaves in question.
  • Figs. 3 - 5 make clear that the transgenic poplar trees created through the process of the present invention exhibit significant toxicity to insects upon ingestion when compared to control plants thus indicating the efficacy of the inserted genetic traits into the trees in question.
  • a plasmid pTV4AMVBTSH has been previously deposited,, at ATCC Accession No. 53636.
  • This plasmid is a cointegrate of two progenitor plasmids, pTV4 and pAMVBTS, as more fully described in published application WO 89/04868.
  • the plasmid pTV4AMVBTSH may be digested with Xho I, a restriction enzyme that separates the two component plasmids in the cointegrate. Religation of the digested DNA under dilute conditions (1-10 microgram/milliliter of DNA) will close the plasmids. If the products are transformed into E. coli and properly selected, the two cointegrates can be isolated.
  • Colonies that are sulfadiazene resistant but ampicillin sensitive will have pTV4 while colonies that are ampicillin resistant but sulfadiazene sensitive will yield the plasmid pAMVBTS. Correct structures may be confirmed by plasmid mini preps.
  • the plasmid pAMVBTS has also been deposited with the ATCC, Accession No. 53637.
  • the plasmid pTVllOO is a cointegrate of progenitor plasmids pTV4 and pCMCHOO.
  • the plasmid pCMCllOO has also been deposited with the ATCC, Accession No. 67641.
  • the plasmid pCMCllOO was derived from pAMVBTS by substituting for the B.t. toxin coding region a coding region for the beta-glucuronidase gene (GUS) .
  • GUS beta-glucuronidase gene
  • DNA of each plasmid may be separately digested with Xho I, the linearized DNA combined, and then the plasmids religated under concentrated DNA conditions (10-50 micrograms/milliliter of DNA) .
  • the desired cointegrate plasmid will be harbored in colonies which are resistant to both sulfadiazene and ampicillin and confirmed by plasmid mini preps.
  • the cointegrates will be of two possible orientations.
  • the desired orientation being the plasmid that has the ligated Xho I site 5 1 to the nopaline synthase promoter of the kanamycin resistance gene (NPT-II or APA-II) from pTV4 to the Xho I site 5' to the CaMV 35s promoter on the GUS gene from pCMCllOO.
  • pAMVBTS which includes a cassette of the CaMV 35s transcriptional promoter, a 5 1 noncoding mRNA leader sequence homologous to the 5• noncoding region of alfalfa mosaic virus coat protein mRNA, a DNA fragment of the first 644 codons of a wild-type B.t. gene from B.t.
  • strain HD-1-Dipel two terminal proline codons, and the nopaline synthase polyadenylation region.
  • a synthetic coding region for this protein has been derived, based on substitution of codons from those found in the native sequence to those which are found most commonly in plant genes. This synthetic sequence was substituted for the first 138 codons of the native sequence from the amino terminus.
  • the entire DNA sequence of plasmid pAMVBT4 is set forth in Fig. 6, where the altered synthetic sequence, which is the only difference between pAMVBTS and pAMVBT4, is between nucleotide 480 (an Nco I recognition site) and nucleotide 897 (a BSP 1286 recognition site) .
  • the synthetic sequence was constructed as six overlapping single stranded oligonucleotides which were annealed into three overlapping duplex strands which were each inserted in turn into pAMVBTS. This procedure may be repeated using pAMVBTS and the sequence of Fig. 6 to make pAMVBT4. This codon substitution has previously been found to enhance the expression of this protein in plant cells.
  • the plasmid pTVBTGUS may be linearized by digestion to completion with the restriction endonuclease Sal I, which cuts pTVllOO at a plasmid unique site between the 3' end of the GUS expression cassette and the 3' end of the gene for ampicillin resistance.
  • the linearized plasmid may then be partially digested with Xho I, which can cut the plasmid in two places, one place being between the NPT-II and GUS genes and the other being immediately adjacent the -26-
  • the desired fragment which is linear DNA of pTVllOO with the ampicillin resistance gene removed, can be purified from the other fragments by agarose gel electrophoresis.
  • the four primary DNA fragments to be expected from this digestion include a full-length pTVllOO with no cuts, fragments cut at the desired Xho I site to detach the ampicillin resistance gene from the GUS cassette, fragments cut at the Xho I site between the GUS cassette and the NPT II cassette, and fragments receiving two cuts at both Xho I sites detaching both the GUS cassette and the ampicillin resistance gene from the NPT II vector.
  • the purified desired fragment will contain the sulfadiazene-resistant vector with the NPT II and GUS cassettes attached, with exposed Sal I and Xho I sites at the ends. Since the exposed ends are compatible in ligation, treatment of this DNA with phosphatase is recommended to avoid self-ligation.
  • the plasmid pAMVBT4 is then digested to completion at a unique Sal I site, located immediately 3 1 to the polyadenylation region of the B.t. toxin expression cassette.
  • the linearized pAMVBT4 may be combined with the phosphatased fragment recovered from pTVllOO, and the two plasmids ligated. If the products are transformed into E. coli and selected for resistance to both sulfadiazene and ampicillin, two alternative plasmids " will be obtained, one being pTVBTGUS and one being a plasmid of the same DNA with the pTVllOO fragment insert being in the opposite orientation. The correct orientation can be identified by plasmid mini prep analysis.

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Abstract

L'invention concerne la création d'arbres transgéniques par un procédé de transformation au moyen de particules. Ce procédé s'est révélé efficace dans le cas de plusieurs types de tissus du peuplier, y compris des protoplastes, des segments internodaux et de pétioles, plus particulièrement dans le cas de cultures de nodosités. On a obtenu des arbres transgéniques qui expriment des niveaux suffisants de la toxine insecticide spécifique du Bacillus thuringiensis, de façon à assurer une toxicité significative contre des insectes qui s'attaquent à ces arbres.
PCT/US1991/003629 1990-05-24 1991-05-23 Transformation d'especes vegetales ligneuses au moyen de particules WO1991018094A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436391A (en) * 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
WO1996025504A1 (fr) * 1995-02-17 1996-08-22 Shell Internationale Research Maatschappij B.V. Modification genetique de vegetaux
EP0784421A1 (fr) * 1994-09-14 1997-07-23 TREETECH MANAGEMENT, INC., doing business as DRY CREEK LABORATORIES Plantes agissant comme pieges pour des organismes nuisibles et protection de plantes cultivees
WO1998000546A2 (fr) * 1996-07-01 1998-01-08 Mycogen Corporation Toxines actives contre les parasites
US6369213B1 (en) 1996-07-01 2002-04-09 Mycogen Corporation Toxins active against pests
WO2016139579A1 (fr) 2015-03-02 2016-09-09 Stora Enso Oyj Production de biomasse

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4795855A (en) * 1985-11-14 1989-01-03 Joanne Fillatti Transformation and foreign gene expression with woody species
WO1989004868A1 (fr) * 1987-11-19 1989-06-01 Agracetus Production de proteines dans des plantes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4795855A (en) * 1985-11-14 1989-01-03 Joanne Fillatti Transformation and foreign gene expression with woody species
WO1989004868A1 (fr) * 1987-11-19 1989-06-01 Agracetus Production de proteines dans des plantes

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Volume 6, No. 8, issued August 1988, MCCABE et al., "Stable Transformation of Soybean (Glycine Max) by Particle Acceleration", pages 923-926. *
HANOVER et al., "Genetic Manipulation of Woody Plants", published 1988, by PLENUM PUBLISHING CORPORATION, see pages 149-166. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Volume 13, Suppl. D, issued 1989, GOLDFARB et al., "Transient Expression of Microprojectile-Introduced DNA in Douglas Fir", page 259. *
NATURE, Volume 328, issued 02 July 1987, VAECK et al., "Transgenic Plants Protected from Insect Attack", pages 33-37. *
PLANT CELL REPORTS, Volume 7, issued 1988, RUSSELL et al., "Recovery of Plants from Leaf Protoplasts of Hybrid-Poplar and Aspen Clones", pages 59-62. *
PLANT PHYSIOLOGY, Volume 89, Suppl. 4, issued April 1989, RUSSELL et al., "Cell Monolayers as an Aid to the Study of Biological Factors Affecting Biolistic Transformation", page 13. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 85, issued November 1988, "Stable Genetic Transformation of Intact Nicotiana Cells by the Particle Bombardment Process", pages 8502-8505. *
TAPPI JOURNAL, Volume 71, No. 7, issued July 1988, A.M. STOMP, "Sex, Designer Genes, and Tree Improvement", pages 115-120. *
WISCONSIN STATE JOURNAL, issued 20 February 1990, R.A. GRIBBLE, "U.W. Scientists Building a Better Tree", pages 1A and 2A. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436391A (en) * 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
EP0784421A1 (fr) * 1994-09-14 1997-07-23 TREETECH MANAGEMENT, INC., doing business as DRY CREEK LABORATORIES Plantes agissant comme pieges pour des organismes nuisibles et protection de plantes cultivees
EP0784421A4 (fr) * 1994-09-14 1998-05-20 Treetech Management Inc Doing Plantes agissant comme pieges pour des organismes nuisibles et protection de plantes cultivees
WO1996025504A1 (fr) * 1995-02-17 1996-08-22 Shell Internationale Research Maatschappij B.V. Modification genetique de vegetaux
WO1998000546A2 (fr) * 1996-07-01 1998-01-08 Mycogen Corporation Toxines actives contre les parasites
WO1998000546A3 (fr) * 1996-07-01 1998-04-09 Mycogen Corp Toxines actives contre les parasites
US6274721B1 (en) 1996-07-01 2001-08-14 Mycogen Corporation Toxins active against pests
US6369213B1 (en) 1996-07-01 2002-04-09 Mycogen Corporation Toxins active against pests
US6752992B2 (en) 1996-07-01 2004-06-22 Mycogen Corporation Toxins active against pests
WO2016139579A1 (fr) 2015-03-02 2016-09-09 Stora Enso Oyj Production de biomasse

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