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WO1993007256A1 - Canon a particules pour introduire de l'adn dans des cellules intactes - Google Patents

Canon a particules pour introduire de l'adn dans des cellules intactes Download PDF

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Publication number
WO1993007256A1
WO1993007256A1 PCT/US1992/008466 US9208466W WO9307256A1 WO 1993007256 A1 WO1993007256 A1 WO 1993007256A1 US 9208466 W US9208466 W US 9208466W WO 9307256 A1 WO9307256 A1 WO 9307256A1
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WO
WIPO (PCT)
Prior art keywords
flying block
opening
cells
chamber
bore
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1992/008466
Other languages
English (en)
Inventor
James Richard Wong
Paul Bellamy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Original Assignee
Ciba Geigy AG
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 Ciba Geigy AG filed Critical Ciba Geigy AG
Publication of WO1993007256A1 publication Critical patent/WO1993007256A1/fr
Anticipated expiration legal-status Critical
Priority to US08/812,215 priority Critical patent/US5955362A/en
Ceased legal-status Critical Current

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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)
    • C12N15/8201—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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • This invention is directed to genetic manipulation of cells and is particularly directed to microinjection of DNA or other genetic material into intact cells.
  • a second guns design uses a different motive force, namely gas generated by electrical discharge through a droplet of water, a different geometry, and a different method of particle preparation.
  • the second gun works at a fixed, very short distance and uses a high voltage electric arc discharge which is hazardous.
  • a third gun uses a timing circuit to meter out a portion of propulsion gas but does not give a reproducible shot
  • a fourth gun is commercially availabe as a supplement to the gunpowder gun.
  • a chamber sealed at one end by a plastic rupture disk is slowly pressurized with compressed gas until the plastic deforms to the point of breakage, whereupon a wave of gas is suddenly released. This wave strikes another plastic sheet which bears the microprojectiles, propelling them towards the target tissue.
  • Particle guns are described in U.S. patent 4,945,050, European patent 270, 356, and Morikawa et al. (1989) Appl. Microbiol. Biotechnol. 31: 320-322.
  • the present invention is directed to a particle gun for the introduction of particles carrying genetic material into intact cells, comprising:
  • an elongated chamber having a principal axis with an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis; a stripper means located on said principal axis between said target holder and said bore opening, said stripper means having an opening therethrough of a cross section smaller than that of said bore portion; a deformable flying block adapted to move along said principal axis in said bore portion while substantially blocking passage of compressed gas past said flying block in said bore portion of said chamber, said flying block being too large to enter said stripper means opening, wherein a surface of said flying block proximal to said opening when said flying block is in said bore is adapted to hold said carrier particles and wherein said flying block is moveable by a compressed gas and yet stoppable on contact of the flying block with said stripper means; means for introducing said flying block into said chamber; an inlet for compressed gas located in said chamber behind said flying block and distally in said bore portion relative to said opening; means for controlling access of
  • the invention also comprises a method for the introduction of genetic material into intact cells, wherein said genetic material, which one desires to have introduced into said intact cells, is prepared, wherein ballistic means are used for accelerating said genetic material at a velocity sufficient to enter into said intact cells and thereby transform them, without damaging said cells, and wherein shoots and whole plants are generated from said transformed cells.
  • the method for the introduction of carrier particles carrying genetic material into intact cells comprises: loading said carrier particles onto a deformable flying block in an elongated chamber having a principal axis and an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis, wherein said flying block is adapted to move along said principal axis in said bore portion while substantially blocking passage of compressed gas past said flying block in said bore portion of said chamber, a stripper means located on said principal axis between said target holder and said bore opening, said stripper means having an opening therethrough of a cross section smaller than said bore portion; said flying block being too large to enter said stripper means opening and a surface of said flying block proximal to said bore opening is adaped to hold said carrier particles and wherein said flying block is moveable by a compressed gas and yet stoppable on contact of said flying block with said stripper means thereby releasing said particles to travel through said stripper means opening into said cells; loading said cells
  • a constant volume of compressed gas can be obtained, for example, by charging a chamber with compressed gas from a source and maintaining said gas at a constant volume and pressure; and charging a reservoir chamber with compressed gas from a source and maintaining said gas at a constant volume and pressure.
  • the method and apparatus of the invention provides for simples particle bombardment having improved economics of operation because the flying block is the only part which is not reused. Replacement of the flying block usually takes only a matter of seconds.
  • the flying blocks are inexpensive and are available with a wide cavity for large particle loads and wide targets, or a narrow cavity for smaller loads and smaller targets.
  • the gun is particularly useful for small targets, such as tissue several millimeters in diameter, in that the particle impact area can be small and the gun can be easily aimed at this area, which is the preferred target for some applications.
  • Figure 1 is a perspective view of one embodiment of a particle gun apparatus of the invention.
  • Figure 2 is a cross-sectional view of a particle gun of the invention, with a schematic diagram of the gas propulsion system.
  • a housing (1) is provided that includes a base plate (14) and a target chamber (25), which can be evacuated to give a vacuum and which is located on the support platform or plate (14).
  • a target holder (12) is mounted in the target chamber (25) and is adapted to hold the sample of living cells, tissue, organs, or embryos (12A) for bombardment by one or more microprojectile particles in order to transfer desirable genetic material into the cells.
  • the microprojectile particles are conventionally known in the ar such as those disclosed in U.S. patent 4,945,050, and are conveniently formed from a metal, such as gold.
  • the particles can be of any size known to those skilled in the art of microinjection via particles but preferably have a diameter from about 0.2 ⁇ m to about 5.0 ⁇ m.
  • the particles are coated or impregnated with the genetic material to be introduced in the manner known in the art
  • the target cells are held on a target holder plate (12) inverted in a jig (2) which can be assembled into the apparatus at variable, discrete distances from die stripper (3) that acts to stop the forward motion of the flying block (17) used to accelerate the microprojectile particles.
  • the target vacuum chamber (25) is typically maintained at a partial, adjustable vacuum during a shot to minimize air resistance and gas blast
  • a vacuum pump (31) is connected to the target vacuum chamber (25) by a vacuum line (36) and is controlled by an electrical switch (5) or other control device.
  • the vacuum line (36) is connected to a pressure gauge (4) and valves (34 and 35) operable to control the pressure.
  • a vent (32) is provided for venting the target vacuum chamber to the atmosphere subsequent to the bombardment of the biological material so that bombarded material can be removed and a new target inserted.
  • the barrel of the gun comprises an elongated chamber (6A) having an opening (30) at one end and an empty bore of invariant cross section.
  • the open end is directed toward a stripper holding plate (16) in a partition (13) that divides the target chamber into two parts, the part distal from the barrel of the gun containing the target holder (12) and the part adjacent to the barrel providing access so that the flying block can leave the gun barrel and strike the stripper that stops forward motion of the flying block (forward being in the direction of the target).
  • This stripper has an opening with a smaller cross section than that of the empty bore of the barrel of the gun and of the flying block.
  • the stripper can be located on and directly attached to the bore or proximally a short distance beyond the bore opening between the bore opening and the target In the latter case, which is preferred, the opening of the stripper is located on the principal axis of the bore.
  • the stripper (3) is in the form of a bolt with an axial passageway oriented so that the flying block strikes the end of the bolt and the microprojectiles pass through the axial passageway on their way to the target
  • the physical shape of the stripper are immaterial to the invention, but a form that can be firmly but removeably attached to the stripper holding plate is preferred so under the impact of shots by the flying blocks. Threads in the stripper with matching threads in the stripper holding plate (as in the described bolt), separate clamping devices, and numerous other techniques can be used to attach the stripper to the stripper holding plate or other device to which the stripper is attached.
  • the stripper is located a short distance from the bore opening in this embodiment which represents a preferred embodiment for easy disposal of used flying blocks, which merely fall to the side and are discarded after striking the stripper.
  • the flying block (17) is the single consumable part of the gun of the invention. It is made of a deformable material, such as a rubber, plastic, deformable metal or the like.
  • a deformable material such as a rubber, plastic, deformable metal or the like.
  • One preferred material is a polycarbonate plastic or similar plastic which absorbs kinetic energy upon striking the stripper via plastic deformation rather than fracturing, thereby eliminating debris.
  • a plastic flying block material can also be formed by injection moding, which is an economical mass production method.
  • a preferred shape has the forward (target) end of the flying block formed to provide a shallow depression or cavity (40) that will hold the microprojectile particles. Different shapes can be provided for this depression to allow for different amounts of particles and different patterns of particle dispersion.
  • the back (breech) end of the flying block can have any shape but is preferably configured for easy handling, for example by providing a slot or cavity that fits a breech pin (see discussion below) to allow easy loading of the block into the particle
  • the stripper (3) can be of any non-deformable material capable of stopping the flying block without significant damage to itself.
  • deformable and non-deformable are relative terms when applied to the flying block and the stripper.
  • an object of the invention is to have a flying block that deforms upon striking the stripper while the stripper remains unchanged by the collision, so that no confusion arises out of the use of these terms that are relative to each other.
  • the stripper is held at a distance from the end of the barrel in a stripper stage or vacuum chamber partition (13), as previously described.
  • the stripper is conveniently a inside-threaded steel cap with an axial passageway smaller than the bore attached to the end of the barrel, which is provided with outside matching threads.
  • the distance from the target to the stripper at which the flying block (17) is stopped can be varied as can be determined by one of skill in the art
  • the flying block (17) is usually stopped at a distance of about 15 mm to about 90 mm, preferably about 25 mm to about 70 mm, from the target
  • Other operating distances can be provided by changing the supporting parts that hold the various working parts, such as the barrel, stripper, and target holder without affecting operation of the particle gun.
  • the elongated barrel chamber (6A) and flying block (17) can be of any geometrical cross-section (relative to the direction of travel of the flying block).
  • the shape of the cross-section is circular, but it can be square, rectangular, or any other shape.
  • the flying block fits the barrel chamber in the same manner as the bullet of a gun, namely with sufficient tightness to prevent significant gas leakage but without being too tight so that movement under the influence of the compressed gas is prevented.
  • the flying clock (17) with the genetic material and particles loaded in the front cavity (40) is introduced into the barrel by carrying the flying block into the breech (6B), typically using forceps; mounting the flying block on a breech bolt pin (6C); sliding the bolt (6E) up into the breech by pushing up in the handle (6D); and rotating the bolt 90 degrees so that the handle engages a slot (6E) which locks the bolt in firing position.
  • the loading mechanism as shown in this embodiment should be familiar, as it is the typical bolt-action loading mechanism of a single shot rifle.
  • the propulsive force on the flying block is provided by a compressed gas.
  • a compressed gas tank (22) or other source of gas is used to charge a reservoir (23) to the desired pressure.
  • the gun provides a controlled, reproducible, adjustable, clean, safe, and inexpensive propulsion source.
  • Single gases such as helium, nitrogen, argon, or carbon dioxide, or mixtures of gases can be used, giving a very large dynamic range for the expansive force of the gas on the rear of the flying block.
  • Hydrogen which has the greatest expansive power, can be used but is not recommended for safety reasons, as it is explosive.
  • the pressure of the compressed gas can vary as can be determined by those of skill in the art
  • the pressure is typically from about 20 psi to about 1500 psi, preferably from about 1000 psi to about 2000 psi.
  • the gas pressure propulsion source of power for firing the flying block at the target has a wide dynamic range, especially when gases with different densities are used, e.g., helium vs. nitrogen vs. carbon dioxide, because the force available from compressed gases at equal pressures varies as the speed of sound through the gas (the speed of sound is higher as the density decreases). Since bottled gas is pure, it also promotes rapid firing cycle times because there is no cleaning between shots as with the prior art use of gunpowder.
  • gases with different densities e.g., helium vs. nitrogen vs. carbon dioxide
  • the gun of the invention incorporates minimum dead volumes in the gas pathway such that the minimum volume for space between valve (20) and back of flying block on the breech pin and balances storage and acceleration volumes (i.e., reservoir and barrel size) in order to obtain optimal capture of the propulsion energy.
  • the volume of the barrel is less than the volume of the reservoir.
  • a pressure regulator (10) and electrically operated solenoid valve (21) are located in line (50) between said gas source and said reservoir (23) for controlling the flow of gas into the reservoir.
  • Pressure gauges (9 and 15) show the pressure of the gas supply (22) and reservoir (23), respectively.
  • a second electrically operated solenoid valve (20) is armed by a first control switch (8) and fired by a second control switch (7).
  • the firing solenoid discharge valve (20) is adapted to discharge the constant volume and pressure gas through line (45) and then through the banjo nut (19) and fitting (18) into the breech (6B) behind the flying block (17) in the embodiment shown.
  • the flying block (17) is thus fired by operation of the firing solenoid discharge valve mechanism.
  • the gas pressure is sufficient to fire the flying block such taht the flying block (17) travels through the bore portion of barrel chamber (6A), carrying the load of DNA-coated particles (17A) in its front cavity (40).
  • the flying block (17) is stopped by the smaller-diameter stripper (3) just past the opening (30) of the bore (6A) in this preferred embodiment. At such time, the particles continue on a straight path to the target cells (12A) while the propulsion gas is diverted to the sides of the barrel (6A). In the embodiment shown, gas is diverted to the sides and away from the target by the stripper and the stripper stage.
  • the deformed flying blocks passage of gas through the suipper; if not diverted this gas could impinge upon and adversely affect the target cells.
  • openings can be provided in the sides of the bore just behind the back end of the flying block when it strikes the stripper nut so that the propulsive gases can escape to the sides. Release of the compressed gas behind the flying block simplifies removal of the flying block after firing and allows further operation of the gun.
  • the target holder bearing the biological target material (12A) is held in a moveable plate on the target stage (12). This arrangement permits rapid, predictable, and precise aiming of particles to within a few millimeters distance on the target holder. For convenience, most of the parts of the apparatus are enclosed in a housing (11) to protect them from dust, moisture, and the like.
  • the used flying block (17) in this preferred embodiment has left the end of the barrel and is thus merely picked up and discarded, an a new flying block is inserted in the breech (6A).
  • the breech bolt (6C) opens and closes with a single motion. No cleaning of the breech is requred because no combustion occurs in the breech.
  • the target tissue (12A) is aligned, the chamber cover (1) is replaced, and the gas reservoir (23) is recharged. The cycle takes about 15 seconds, as compared to several minutes in the prior art devices.
  • The.entire device can be oriented to fire the flying block any desired direction, such as upward, downward, or to the side as is most convenient for example, to simplify holding a variety of target cells in position.
  • the particle gun of the invention has been used to obtain transient expression of introduced nucleic acids, i.e. temporary genetic transformation, in several plant cells and tissues such as tobacco, wheat corn, barley, oat onion, and sorghum, and stable transformation of tobacco, wheat and corn cells.
  • This ⁇ -Glucuronidase (GUS) expression vector was derived from plasmid pBl 121, purchased from Cloneteck Laboratories, Palo Alto, Califormia. Intron 6 of the maize Adhl gene was amplified by PCR from plasmid pB428, described in "Bennetzen et al., Proc. Natl. Acad. Scl, USA 81:4125-4128 (1987)", using oligonucleotide primers SON0003 and SON0004.
  • SON0003 5'-CTCGGATCCAGCAGATTCGAAGAAGGTACAG-3'
  • SON0004 5'-ACGGGATCCAAC ⁇ CCTAGCTGAAAAATGGG-3'
  • the PCR reaction product was digested with restriction endonuclease BamHl, cleaving the BamHl site added on the 5' end of each PCR primer.
  • the resulting DNA fragment was purified on an agarose gel and ligated into the BamHl site of pB1121, which is between the CaMV 35S promoter and the CUS gene.
  • the ligated DNA was transformed into E.coli and clones with the Adhl intron 6 in the same orientation as the GUS gene were identified by restriction digest
  • DHFR dihydrofolate reductase
  • the resulting fragment was digested with restriction endonucleases Pstl and BspHl and purified on an agarose gel.
  • the DHFR coding region was produced by PCR amplification of pHCO using primers SON0016 and SON0017.
  • SON0016 5'-GCTACCNrGGCCACATAGAACACC-3'
  • SON0017 5'-CGAGAGCTCCiCAC ⁇ CAACC ⁇ G-3'
  • the resulting fragment was digested with restriction endonucleases Nsol and Sacl and purified on an agarose gel.
  • the two fragments described above were ligated into a vector fragment prepared from pB1121 by digestion with restriction endonucleases Pstl and Sacl and purification of die 3 kb fragment containing the Nos terminator region and pUC19 region of pB1121 on an agarose gel.
  • This three way ligation fused the 35S promoter- Adhl intron 6-DHFR gene-Nos terminator in correct order and orientation for functional expression in plants.
  • This GUS expression vector was derived from pSOG 10 by the insertion of the maize chlorotic mottle virus (MCMV) leader, described in "Lommel et al., Virology 181: 382-385 (1991)", into the 35S-GUS gene non-translated leader by a three way ligation.
  • MCMV maize chlorotic mottle virus
  • Both strands of the 17 bp MCMV capsid protein leader sequence plus appropriate restriction endonuclease sights were syntiiesized and annealed.
  • the resulting double stranded fragment was degested with BamHl and Ncol and purified on an acrylamide gel.
  • the GUS gene coding region was amplified by PCR using primers SON0039 and SON0041 and pB1121 as a template.
  • SON0039 5'-CGACATGGTACGTCCTGTAGAAACCCACA-3'
  • SON0041 5'-ATCGCAAGACCGGCAACAGGATTC-3'
  • the GUS gene eas removed from the plasmid pSOG 10 by digestion with restriction endonuclease Sacl and partial digestion with restriction endonuclease BamHl.
  • the resulting vector which has a BamHl site and a Sacl site in which to reinsert a coding region behind the 35S promoter-Adhl intron 6, was purified on an agarose gel.
  • the three fragments described above were ligated in a three way ligation to produce a gene fusion with the structure: 35S promoter-Adhl intron 6-MCMV leader-GUS-Nos terminator, all in the pUC 19 vector backbone.
  • the DHFR selectable marker vector is identical to pSOG 19, except that the MCMV leader is inserted in the non-translated leader of the DHFR gene to enhance translation. It was created in two steps. First the GUS coding region in pSOG 32, a vector identical to pSOG 30 except that it contains a modified Adh promoter rather than 35S, was replaced with DHFR coding region from pSOG 19 by excising the GUS with Ncol and Sacl and ligating in the DHFR as an Ncol-Sacl fragment This resulting in vector pSOG 33 which has the gene structure Adh promoter-Adhl intron 6-MCMV leader-DHFR coding region-Nos terminator, with a Bglll site between the promoter and Intron and a Sacl site between the coding region and the terminator.
  • the Bglll-Sacl fragment was isolated by restriction endonuclease digestion and agarose gel purification, and ligated into the BamHl and Sacl sites of pSOG 30, replacing the Adhl i ⁇ tronZ6-MCMV leader-GUS coding region of pSOG30 with the Adhl intronZ6-MCMV leader-DHFR coding region of pSOG33.
  • Callus cultures are maintained on IMS medium, as described for example by "Murashige, T. and Skoog F., 1962, Physiol. Plant 15, 473 - 497” (MS salts, vitamins, iron, 3% sucrose, 0.7% agar, 1 mg/liter 2,4-D).
  • the suitable callus cultures comprise among others Type ⁇ callus (a friable and embryogenic type of callus) obtained from “shoot-competent" cell cultures as described in "W. Wang and H. Nguyen, 1990, Plant Cell Reports 8639-642”) after recurrent subculture and visual selection. They are subcultured every two weeks and are kept in the dark at 26°C. Suspension cultures are maintained in IMS liquid medium and are subcultured twice weekly. They are kept in the dark at 26°C and shaken at ⁇ 125 rpm.
  • Cell Preparation for Bombardment The cells are given a plasmolysis treatment before bombardment. Packed cell volume is measured and cells are diluted in IMS liquid medium with added osmoticum: 0.4M sorbitol for suspension cells and 0.6M sorbitol for callus cells. Cells are diluted such that the final packed cell volume per target is 1/20 ml for a fine suspension and 1/10 ml for callus. Diluted cells are placed in a 250 ml flask containing a stir bar and are stirred for a minimum of 30 minutes, up to a few hours.
  • Gold particles (1.0 micron; from Bio-Rad) are washed by aliquoting into a microfuge tube, adding ⁇ 1 ml 100% ethanol, vortexing, spinning down, removing die supernatant, and repeating twice with sterile water. After the final wash, as much water is removed as possible and polylysine solution (0.02% polylysine + 15 mM ammonium acetate) is added to completely immerse the particles. The particles are vortexed, spun, and the supernatant removed. The particles are allowed to dry overnight in a laminar flow hoodor for 30 minutes under a gentle nitrogen stream.
  • a "full" particle preparation For a "full" particle preparation, weigh out 10 mg particles and place in sterile microfuge tube containing a stir bar. Add 100 ⁇ l (1 ⁇ g/ ⁇ l) DNA (according to step I), vortex, add 10 ⁇ l 100 mM Na 2 HPO 4 , vortex, add 10 ⁇ l 100 mM CaCl 2 , vortex, add 380 ⁇ l 100% ethanol, vortex. Stir suspension vigorously on stir plate while pipetting 3 ⁇ l onto each plastic flier (projectile). Allow particles to dry onto fliers for at least 15 minutes before bombarding.
  • Colonies that continue to proliferate for about 3 - 4 weeks are then transferred to "0.5MS" maintenance medium: MS salts, vitamins, iron, 3% sucrose, 0.7% agar, 0.5 mg/liter 2,4-D. Tissue is subcultured onto tiiis medium biweekly until embryogenic structures appear or tissue seems suitable for regeneration.
  • Tissue is transferred to MS medium containing either 3 mg/liter BAP or 1 mg/liter NAA + 5 mg/liter GA, and plates are moved to the light After 2 - 4 weeks, tissue is transferred to MS medium without hormones. Shoots that appear are placed in Magenta boxes containing either MS medium without hormones or MS medium with 0.5 mg/liter NAA. When sufficient root and shoot growth has occurred, plantlets are transferred to soil and placed in a phytotron.

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Abstract

Un canon à particules pour introduire du matériau génétique dans des cellules, tissus ou organes utilise pour propulser les particules une propulsion à gaz depuis un réservoir de gaz comprimé. D'autres caractéristiques des modes préférés de réalisation incluent un élément de séparation qui arrête, à l'extérieur de l'âme du canon, le bloc porteur de particules, facilitant ainsi le rechargement. Un procédé d'utilisation du canon est également décrit.
PCT/US1992/008466 1991-10-07 1992-10-05 Canon a particules pour introduire de l'adn dans des cellules intactes Ceased WO1993007256A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US08/812,215 US5955362A (en) 1991-10-07 1997-03-06 Methods for stable transformation of wheat

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US77243591A 1991-10-07 1991-10-07
US772,435 1991-10-07

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US77243591A Continuation-In-Part 1991-10-07 1991-10-07

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US99239192A Continuation-In-Part 1991-10-07 1992-12-16

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WO1993007256A1 true WO1993007256A1 (fr) 1993-04-15

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PCT/US1992/008466 Ceased WO1993007256A1 (fr) 1991-10-07 1992-10-05 Canon a particules pour introduire de l'adn dans des cellules intactes

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AU (1) AU2781892A (fr)
WO (1) WO1993007256A1 (fr)

Cited By (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013822A3 (fr) * 1992-12-16 1994-09-15 Ciba Geigy Ag Procede de transformation stable de ble
WO2003014357A1 (fr) 2001-08-09 2003-02-20 University Of Saskatchewan Plants de ble presentant une resistance accrue aux herbicides a base d'imidazolinone
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US11499162B2 (en) 2016-07-15 2022-11-15 Basf Se Plants having increased tolerance to herbicides
US11149030B2 (en) 2016-07-27 2021-10-19 BASF Agro B.V. Plants having increased tolerance to herbicides
WO2018019860A1 (fr) 2016-07-27 2018-02-01 BASF Agro B.V. Plantes ayant une tolérance accrue aux herbicides
US11879132B2 (en) 2016-12-20 2024-01-23 BASF Agro B.V. Plants having increased tolerance to herbicides
WO2018114759A1 (fr) 2016-12-20 2018-06-28 BASF Agro B.V. Plantes ayant une tolérance accrue aux herbicides
US11479786B2 (en) 2017-11-29 2022-10-25 Basf Se Plants having increased tolerance to herbicides
US12123010B2 (en) 2017-11-29 2024-10-22 Basf Se Plants having increased tolerance to herbicides
US11939587B2 (en) 2018-01-17 2024-03-26 Basf Se Plants having increased tolerance to herbicides
WO2023031161A1 (fr) 2021-09-03 2023-03-09 BASF Agricultural Solutions Seed US LLC Plantes présentant une tolérance accrue aux herbicides
WO2024126113A1 (fr) 2022-12-12 2024-06-20 BASF Agricultural Solutions Seed US LLC Plantes ayant une tolérance accrue aux herbicides
WO2025153595A1 (fr) 2024-01-17 2025-07-24 Basf Se Plantes présentant une tolérance accrue aux herbicides
WO2025153657A2 (fr) 2024-01-17 2025-07-24 Basf Se Plantes ayant une tolérance accrue à des herbicides

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