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WO1994000583A1 - Transformation de plantes par injection directe d'adn____________ - Google Patents

Transformation de plantes par injection directe d'adn____________ Download PDF

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Publication number
WO1994000583A1
WO1994000583A1 PCT/US1993/005825 US9305825W WO9400583A1 WO 1994000583 A1 WO1994000583 A1 WO 1994000583A1 US 9305825 W US9305825 W US 9305825W WO 9400583 A1 WO9400583 A1 WO 9400583A1
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Prior art keywords
dna
plant
ovule
embryo
plants
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PCT/US1993/005825
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English (en)
Inventor
Anne M. Espinasse-Gellner
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South Dakota State University
University of South Dakota
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South Dakota State University
University of South Dakota
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Priority to AU45395/93A priority Critical patent/AU4539593A/en
Publication of WO1994000583A1 publication Critical patent/WO1994000583A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—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

Definitions

  • This invention relates to transformation of plants, including sunflower, Helianthus annuus L. by direct injection of DNA into a fertilized ovule at the onset of embryogenesis.
  • Microprojectile bombardment to introduce alien DNA into the genome of a host plant is exemplified by Dupont' ⁇ Biolostic Particle Delivery System. [See also e.g. Sanford et al., Part. Sci. and Techn., 5, 27 (1987); European Patent Application No. 331,855 Sanford et al.]
  • Glycine max L. bombardment of over 20,000 shoot-apices only resulted in transient expression of the transforming gene whereas bombardment of embryogenic suspensions with the same gene produced several transformed plants (Buising et al., Proc. 3rd Biennal Conf. on Molecular and Cellular Bio, of the Soybean, July 1990; Finer and McMullen, Proc. 3rd Biennal Conf. on Molecular and Cellular Bio, of the Soybean, July 1990).
  • the present invention relates to a method for transformation of plants by direct injection of exogenous DNA into the endosperm of a fertilized plant ovule at the onset of embryogenesis.
  • the exogenous DNA sequence will correspond to a desired physical or functional characteristic to be imparted to the host plant.
  • the DNA sequence is introgressed into the host plant genome, thus producing a transformed plant embryo.
  • DNA can be injected into a fertilized plant ovule at a preglobular stage, globular stage, or heart-shaped stage of plant embryos.
  • Example times for injection of exogenous DNA into a fertilized plant ovule is at about 12-96 hours after host plant fertilization.
  • the fertilized plant ovules are cultured on a growth medium to develop embryos capable of growing into transformed plants on the same media.
  • these ovules are positioned horizontally on the growth medium employed for culturing.
  • injection of exogenous DNA is accomplished using a tapered needle that is inserted into the endosperm through the butt end of the ovule, about two-thirds of the way through the ovule towards the embryo.
  • an approximately 1 ⁇ l plasmid solution having about 1 ⁇ g/ ⁇ l to 1 ng/ ⁇ l of plasmid DNA is injected into the endosperm of the host plant ovule.
  • the method of the present invention is particularly well suited for transformation of sunflower, Helianthus annuus L.
  • the present invention is believed to be applicable to dicotyledonous and monocotyledonous plants in general and could be employed for direct transformation of wheat, soybean and corn ovules, for example.
  • the method of the present invention provides for direct transfer of exogenous DNA to a host plant ovule at the onset of embryogenesis and in vitro culturing of ovules that produces transformed plants without the need for a vector or the regeneration of plants from cultured explants.
  • Fig. 2 shows autoradiograms showing presence of plasmid pBI221 in leaf DNA of putative transgenic plants (T) [P: control plasmid].
  • Fig. 3 shows histochemical identification of the GUS-gene in putative transgenic plants (blue spots).
  • Fig. 4 shows electrophoresis and autoradiograms of PCR products from DNA amplification of transgenic plant progenies [lane 1: 1 kb DNA ladder].
  • the present invention is directed to a method for producing transformed plants, including dicotyledonous and monocotyledonous plants including all species of sunflowers, e.g. Helianthus annuus L., soybeans, e.g. Glycine max L. , corn, e.g. Zea mays, wheat, e.g. Triticum aestivum or the like.
  • the present invention provides for the direct transfer of exogenous DNA to host plants and integration of the exogenous DNA into the host plant genome. Progenies of the transgenic plants inherit the extrachromosomal DNA.
  • heterologous, exogenous, or foreign DNA refers to DNA originating from a source outside the host or recipient plant.
  • DNA segment refers to a DNA sequence having a nucleotide base composition and length capable of being introgressed into the genome or a gene complex of a host plant.
  • host plant refers to a plant chosen to receive an exogenous DNA segment.
  • gene refers to a segment of DNA composed of a transcribed region and a regulatory sequence that makes transcription possible and can, either alone or in combination with other genes, provide the organism with an identifiable trait.
  • trait refers to detectable physical or functional characteristic of an organism.
  • phenotype is a particular manifestation of a trait which corresponds to the presence of a particular gene.
  • homoology refers to two regions of DNA which contain regions of nearly identical DNA sequences.
  • Transformant refers to a host plant which has been transformed with genetic material according to the methodology of the invention.
  • transgenic is used herein to include any plant, plantlet, or fertilized ovule the genotype of which has been altered by the presence of "heterologous, exogenous, or foreign DNA,” wherein the DNA was introduced into the genome by the described genetic engineering process according to the invention, or which was initially introduced into the genome of a parent plant by the process of the present invention and is subsequently transferred to later generations by sexual crosses or asexual propagation.
  • gene refers to the sum total of hereditary genetic material within a cell's chromosomes.
  • the term “heritable” means that the DNA is capable of transmission through at least one complete sexual cycle of a plant, i.e., it is passed from one plant through its gametes to its progeny plants.
  • plasmid refers to autonomously replicating extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature.
  • the method of this invention produces transformed plants by (i) introducing exogenous genetic material into a fertilized plant ovule of a host plant at the onset of embryogenesis in a manner providing for introgression of the genetic material into the genome of the fertilized ovule of the host plant, and (ii) culturing the fertilized ovule on growth medium to maximize generation of transformed plantlets capable of developing into transformed plants which themselves can transmit to their progeny a trait resulting from the presence of the exogenous DNA in the host plant genome.
  • Plant Species and Development Stage for Transformation Process and Embryogenesis The method of the invention is believed to be applicable to a wide range of plants including species of wheat, soybean, corn, or the like. The method is particularly useful to produce transformed sunflower plants.
  • introduction of an exogenous DNA segment is made at the onset of embryogenesis inside a fertilized plant ovule. It is believed that one of skill in the art will recognize the onset of embryogenesis of a fertilized ovule in plants. For sunflower, this occurs from about 4 hours after fertilization and extends until about 24 hours when the fertilized ovule begins to swell and darken.
  • the method of the invention can be employed when the fertilized ovule is in the preglobular, globular or heart-shaped stage of embryo development.
  • exogenous DNA introduction is preferably made from about 12 hours to about 96 hours after ovule fertilization. It is to be understood that globular and heart-shaped stages of development may be reached faster or slower depending on the particular plant or crop, and growing conditions in the greenhouse or growth chamber.
  • the method can be used to introduce exogenous DNA into ovules having embryos not yet visible with the naked eye or dissecting scope.
  • the method is more effective when DNA is introduced into ovules in which the embryo is somewhat visible with a dissecting scope; that is, globular stage (less than about 0.1 mm) or when the DNA is introduced into ovules in which the embryos are visible with the dissecting scope; that is, heart-shaped stage (0.1-0.5 mm) .
  • the exogenous DNA that is introgressed into the host plant genome can be derived or isolated from any source, and may be chemically altered prior to introduction into a fertilized plant ovule.
  • the exogenous DNA can be an identified useful fragment from a plant other than the host plant, and which is chemically synthesized in pure form.
  • the DNA of interest can be removed from the source of interest by chemical means such as using restriction endonuclease, so that it can be further modified if desired by amplification and methodology of genetic engineering.
  • the exogenous DNA can be completely synthetic DNA, semi-synthetic DNA, DNA isolated from non-host plant biological sources, or the like.
  • the exogenous DNA includes but is not limited to DNA from plant genes, and non-plant genes such as those from bacteria, yeasts, animals or viruses, modified genes, portions of genes, and chimeric genes of diverse origin including plants other than the host plant.
  • the exogenous DNA used for transformation herein may be circular or linear, double-stranded or single-stranded.
  • the DNA is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences which promote the expression of the exogenous DNA present in the transformed plant.
  • the exogenous DNA may itself comprise or consist of a promoter that is active in the host plant or may utilize a promoter already present in the host plant genome.
  • the compositions of, and methods for, constructing DNA segments which can transform certain plants are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful in the invention.
  • the present invention is not directed to the specific exogenous DNA and is not dependent upon the exogenous DNA composition used.
  • the exogenous DNA will in many cases be a plasmid-borne gene for a recognizable phenotypic trait.
  • suitable DNA segments for use in the present invention include selectable marker genes, reporter genes, enhancers, introns, and the like.
  • the exogenous DNA can be a sequence that expresses, inhibits, enhances, or modifies biological activity when introgressed into the host plant genome and when coupled to an appropriate targeting sequence such as a homologous insertion sequence. Suitable methods of construction for exogenous DNA segments are provided in the literature [See J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed. , 1989)].
  • the exogenous DNA will be relatively small such as less than about 30 kb. Relatively short sequences are preferred because they minimize susceptibility to physical, chemical, or enzymatic degradation which increases as the size of the DNA increases.
  • Exogenous DNA useful in the invention includes DNA which provides for, or enhances, a beneficial feature of the resultant transformed plant.
  • the DNA may encode proteins or antisense DNA transcripts in order to promote increased food values, (e.g. increased essential amino acid content) higher yields (increased lysine production), pest resistance (e.g. Bacillus thurinqiensis, endotoxin) , disease resistance, herbicide resistance, and the like or to modify the host plant physiology (reduction of flower abortion in soybean) .
  • Microinjection of DNA An important aspect of the invention is the manner for delivery of exogenous DNA into the fertilized plant ovule. Unlike known techniques of plant transformation, the invention employs microinjection of exogenous DNA into the endosperm of a fertilized plant ovule.
  • the DNA is delivered, preferably through a small volume (e.g., less than 5 ⁇ l) capillary tube that has a needle-like tapered end, preferably about 1 ⁇ l [about 0.04 mm].
  • the exogenous DNA is preferably in a biologically suitable solution, such as TE(10 mM Tris- HCl pH 8.0/1 mM EDTA pH 8.0).
  • the amount of plasmid in solution can vary from about 2 ⁇ g/ ⁇ l to 1 ng/ ⁇ l.
  • the pressure applied during DNA injection and the pressure within the ovule must be appropriate to accomplish exogenous DNA incorporation.
  • the amount of DNA solution injected into the ovule should be between about 15 percent to 30 percent of the volume of that ovule.
  • the volume of exogenous DNA- containing solution should be about 1 ⁇ l for sunflower ovules. While the amount of exogenous DNA can vary the amount of exogenous DNA solution should not increase the volume of the ovule excessively to avoid rupture.
  • the invention involves exogenous DNA injection into the fertilized ovule endosperm.
  • the injection is made from the butt end of the ovule opposite the micropyle end. It has also been found preferable to insert the needle through the butt end about two-thirds of the way into the ovule through the area of the endosperm membrane/megasporangium, and into the endosperm. Embryo Culturing and Growth
  • the developing embryos are preferably cultured in vitro on a suitable growth medium.
  • the growth medium will of course vary depending on the particular plant. It is envisioned that the medium composition will be standard growth medium as understood by those of skill in the art. For example, the following can be used: salt solutions of Murashige and Skoog rPhysiol. Plant, 15, 473-497 (1962)], Gamborg et al. rExp. Cell Res.
  • fertilized ovules be horizontally positioned on the medium during in vitro culturing.
  • the time and temperature for culturing the fertilized ovules will also vary depending on the plant. In the case of sunflower, fertilized ovules will be grown for about two weeks at about 25°C under about 16 hours of light per day.
  • plant embryos are extracted from the ovule and grown on fresh growth media of the same composition into plantlets. Plantlets are subsequently transferred to pots and grown to maturity. Some seeds from mature transformed plants possess the heritable genetic information corresponding to the exogenous DNA introduced to the fertilized host plant ovule.
  • Evidence of host plant transformation and introgression of exogenous DNA can be determined using known histological, molecular, and immunological techniques. Such techniques as understood by those skilled in the art are represented by the techniques employed in the Example.
  • Transformed plants produced by the method of the invention are expected to be useful for a variety of commercial and research purposes.
  • Transformed plants can be created for use in traditional agriculture to possess traits beneficial to the grower (e.g., agronomic traits such as pest resistance, herbicide resistance or increased yield) , beneficial to the consumer of the grain harvested from the plant (e.g., improved nutritive content in human food or animal feed) , or beneficial to the food processor (e.g., improved processing traits).
  • Transgenic plants may also find use in the commercial manufacture of proteins or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like. Cells or tissues from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules.
  • Two specific uses of the invention are to supplement the nutritional value of sunflower by introgressing genetic material for soybean seed storage proteins into sunflower and increase sunflower genetic resistance to insects by introgressing toxin genes into sunflower.
  • Ovule and Embryo Culture Ovaries were collected in March for the January planting (series I) and in June/July for the April planting (series II) for two days after hand- pollination, and surface-sterilized 10 minutes in a 40% commercial bleach solution with Tween 80 (12 drops for 200 ml).
  • hand-pollination with fresh pollen of RHA 297 and RHA 299 was done in the afternoon twice when stigmata of female lines were receptive. Three collecting times were employed: I: 16 to 20 hours after hand-pollination;
  • Ovaries were darkening (grayish rather than whitish) , swollen and with thickened walls. These characteristics indicate that ovaries have been fertilized and that their ovules carried one embryo.
  • Embryo development in collected ovaries was as follows: Stage I ovaries: Embryos not visible with naked eye or dissecting scope. They were at best globular.
  • Stage II ovaries Embryos somewhat visible with a dissecting scope. They were at the globular stage (about 0.1 mm) .
  • Stage III ovaries: Embryos visible with a dissecting scope. They were at the heart-shaped stage (0.1-0.5 mm). Ovules (20-75) were excised aseptically and planted on three different media in 15 x 100 mm petri dishes sealed with parafilm. These media were MS (Murashige and Skoog, supra, 1962), B5 (Gamborg et al., supra, 1968), NN (Nitsch and Nitsch, supra, 1969) supplemented with 0.1 mg/1 naphthalene acetic acid and 3% sucrose, and solidified with 0.8% agar. Both treated and control ovules were grown for 14 days on the three media under 16-hour cool white light and 25°C ⁇ 2°C.
  • Ovules were then opened, developing embryos were excised and grown on the same fresh medium for germination into small plantlets. Plantlets were transferred onto the same media in baby food jars and greenhouse planted in pots in commercial Sunshine mix when both shoots and roots were sufficiently developed (3 to 5 cm long). Seeds were recovered from most plants upon selfing. The results are for the three media confounded.
  • Plasmid pBl221 was purchased from CloneTech (Palo Alto, CA) (CloneTech 1992/1993 Catalog, p. 174, and GUS Gene Fusion System User's Manual, incorporated by reference herein) . It contains a 3 kb insert composed of the CaMV 35S promotor, /3-glucuronidase gene (GUS gene), and Nopaline Synthase polyadenylation region on a Hindlll-EcoRl fragment of pUC19.
  • the plasmid was amplified in E_;_ coli.:DH10BTM [purchased from Bethesda Research Laboratories (BRL), Gaithersburg, MD] according to the manufacturer procedure. More specifically, this procedure involved the following materials and protocol:
  • MATERIAL E. coli: DH10BTM competent cells from BRL This strain has a high efficiency of transformation and carries the mcrBC " and mrr ⁇ deletions that minimize elimination of inserted methylated DNA (stored at -70°C) .
  • Bactotryptone 2 g Yeast Extract 0.5 g NaCl 1 ml from a 1 M stock solution (5.844 g/l00 ml) KC1 0.25 ml from a 1 M stock solution (7.455 g/l00 ml) Millipore water 99 ml
  • Collect one colony from streak plate to inoculate 5 ml LB + ampicillin broth (optimum 1 colony for 10 ml of medium) Grow suspension at 37°C from 6 hrs. to overnight under shaking conditions Pipet 4 x 1 ml of that suspension to inoculate 4 x 250 ml of LB + ampicillin in 1000 ml flask (optimum culture volume to flask volume: 1:10 to 1:30)
  • the pBI221 plasmid was extracted according to the Alkaline extraction procedure stated in Berger and Kimmel, Methods in Enzymology, 152, 166-167 (1987), incorporated by reference herein. This procedure employed:
  • Pellet transformed bacteria by centrifuging liquid culture 10 min. at 5000 rpm (use centrifuge bottles, do not exceed 5000 rpm)
  • the pBl221 plasmid was then purified by EtBr/CsCl density gradient using the procedure of Berger and Kimmel, supra (1987), incorporated by reference herein. This procedure involved:
  • the needle used to inject ovules was a 3 ⁇ l handmade needle made from a 5 ⁇ l capillary tube that was heated on an alcohol lamp, then drawn thinner under a dissecting microscope. The needle was inserted into the butt end of the ovule, two-thirds straight inside the ovule, to reach the endosperm-containing embryo sac of the ovule. Where the needle is located can be seen by transparency in the light.
  • the plasmid solution is delivered into the endosperm containing "embryo sac" of the ovule using a wire plunger that pushed the solution inside. Only 1 ⁇ l of plasmid solution was injected. Larger amounts (e.g.. 2 ⁇ l) caused the ovule to burst. Using this method a large number of ovules (400 or greater) can be plated and treated in a working day by an individual.
  • Ovules were laid down, that is horizontally positioned on top of the media.
  • the culture media used were the salt solutions of Murashige and Skoog, supra (1962), of Gamborg et al., supra (1968), and of Nitsch and Nitsch, supra (1969) supplemented with 3% sucrose, 0.1 mg/L nephtalene acetic acid, Gamborg's vitamins and 0.8% agar, each available from Sigma, St. Louis, MO as Murashige and Skoog Based Media formula M0404, Gamborg's Based Media G5893, and Nitsch and Nitsch Media N5639, pages 127, 131, and 137 of Sigma Catalog 90, incorporated by reference herein.
  • Standard growing conditions were used. These were 16 hours cool white light and 25°C.
  • Second Series 1857 ovules were plated - 891 embryos were recovered - 68 developed into viable plantlets - 29 flowering plants were obtained including 11 control and 18 putatively transformed plants.
  • Extraction of -glucuronidase was performed according to the protocol provided by CloneTech in the GUS Gene Fusion Systems User's Manual, pp. 1-5, incorporated by reference herein. This procedure employed a lysis buffer and grinding with a polytron. Identification employed a goat anti-rabbit alkaline phosphatase conjugate as secondary antibody of the rabbit anti-glucuronidase serum. Specifically, leaf extracts from putative transgenic and control plants were blotted on a nitrocellulose membrane and washed with 40 ml TSW buffer for one hour. TSW buffer comprised 10 mM Tris-Cl pH 7.4, 0.9% NaCl, 0.25% carnation instant non-fat milk, 0.1% Triton X-100, and 0.02% SDS.
  • TSW buffer was then replaced with AP development buffer [33 ⁇ l NBT (nitro blue tetrazolium) 50 mg/ml in 90% dimethyl formamide and 16.5 ⁇ l BCIP 5-bromo-4-chloro-3-indolyl-phosphate 50 mg/ml in dimethylformamide was added] and allowed to develop for 20-30 min. Purple coloration indicates presence of 3-glucuronidase. Results of the test performed on plants derived from series II ovules are given in Figure 1.
  • Leaf-DNA from putatively transformed and control plants was extracted according to Doyle and Doyle's technique (Focus 12(1) p. 13-15) modified as follows: an extra grinding was added after grinding in liquid nitrogen using a polytron. RNAs were eliminated using LiCl precipitation rather than RNase.
  • Extracted DNA was digested by Hind III and EcoRI according to standard procedure (Current Protocols in Molecular Biology, Ausubel et al. (1987), incorporated by reference herein) .
  • the surface of the sponge, when wet, should be larger than the gel.
  • the membrane Upon drying, the membrane will assume its characteristic curl.
  • To place membrane into the hybridization bag manually uncurl one end and slide this end into the bag. Once this end is inside the bag, gently slide the rest of the membrane into the bag.
  • Membranes were hybridized for 24 hours with 32 p-labelled pBI221 plasmid.
  • the probe was prepared using Pharmacia Oligolabelling Kit and the Klenow fragment according to manufacturer instructions provided with the product. The procedure involves:
  • Controls were the doubled digested plasmid (by HindiII and EcoRI), the ⁇ ladder and the control plants (injected with TE).
  • Plantlets from each series that tested positive in one or more tests are listed in Table I. Differences in responses among tests may be due to the chimeric character of the transformed plants. Although plasmid PBI221 was injected as early as possible following fertilization inside the ovules, the developing embryos may have already included several cells and only one cell may have integrated the foreign DNA, thus producing a chimeric transgenic plant. However, these tests established that a total of 11 plants out of 34 for the two series were at least partially transgenic (tested positive in two or three tests). This corresponds to a 30% averaged success rate across both series.
  • Table I Plants from series I and II that tested positive in one or more of the GUS assays.
  • Table II Number of progenies derived through open pollination from confirmed transgenic plants testing positive for the GUS-gene using the PCR technique.
  • transgenic lines of sunflower were derived from the application of the technique of transformation according to the invention and described herein.

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Abstract

Une technique de micro-injection permet la transformation directe de plantes. Le procédé utilisé implique l'injection d'ADN exogène dans l'ovule fertilisé d'une plante lors du démarrage de l'embryogénèse.
PCT/US1993/005825 1992-06-23 1993-06-17 Transformation de plantes par injection directe d'adn____________ Ceased WO1994000583A1 (fr)

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AU45395/93A AU4539593A (en) 1992-06-23 1993-06-17 Transformation of plants by direct injection of dna

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US90320592A 1992-06-23 1992-06-23
US07/903,205 1992-06-23

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WO1998001575A1 (fr) * 1996-07-08 1998-01-15 Pioneer Hi-Bred International, Inc. Transformation de cellules de zygote, d'oeuf et de sperme et recuperation de plantes transformees dans des sacs embryonnaires isoles
WO2001059140A1 (fr) 2000-02-09 2001-08-16 The University Of York Cellules transgeniques exprimant des acides nucleiques de glucosyltransferase
US7026528B2 (en) 1996-06-21 2006-04-11 Monsanto Technology Llc Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom
WO2008142364A2 (fr) 2007-05-22 2008-11-27 Plant Bioscience Limited Composition et methode de modulation du developpement du chevelu racinaire de plantes
EP2036984A2 (fr) 2002-07-26 2009-03-18 BASF Plant Science GmbH Reversion de l'effet sélectif négatif d'un protéin de marquage comme procédure de sélection
EP2045327A2 (fr) 2005-03-08 2009-04-08 BASF Plant Science GmbH Expression à amélioration de séquences d'intron
EP2159289A2 (fr) 2005-06-23 2010-03-03 BASF Plant Science GmbH Procédés améliorés pour la production de plants stablement transformés
EP2163635A1 (fr) 2004-08-02 2010-03-17 BASF Plant Science GmbH Procédé d'isolation de séquence de terminaison de transcription
EP2184363A1 (fr) 2003-11-10 2010-05-12 Icon Genetics GmbH Système d'expression dans les plantes dérivé d'un virus a ARN
WO2010061187A2 (fr) 2008-11-25 2010-06-03 Algentech Sas Procédé de transformation de mitochondries de plante
WO2010061186A2 (fr) 2008-11-25 2010-06-03 Algentech Sas Procédé de transformation de plastide de plante
WO2010084331A2 (fr) 2009-01-26 2010-07-29 Algentech Sas Ciblage de gène dans des plantes
WO2010146359A1 (fr) 2009-06-15 2010-12-23 Plant Bioscience Limited Production de capsides virales
WO2011017288A1 (fr) 2009-08-05 2011-02-10 Chromocell Corporation Plantes, microbes et organismes enrichis
EP2336335A2 (fr) 2004-07-07 2011-06-22 Icon Genetics GmbH Procédé d'expression transitoire de protéines dans les plantes biologiquement sûr
EP2357239A1 (fr) 2009-10-29 2011-08-17 Universität zu Köln Procédés et moyens pour un système marqueur sélectionnable dans les plantes
WO2011148135A1 (fr) 2010-05-25 2011-12-01 The University Court Of The University Of Aberdeen Translocation à travers des membranes cellulaires eucaryotes en fonction de motifs de séquences de protéines d'oomycète
US8153863B2 (en) 2007-03-23 2012-04-10 New York University Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity
WO2012058762A1 (fr) 2010-11-04 2012-05-10 Medicago Inc. Système d'expression végétale
US8790928B2 (en) 2003-07-31 2014-07-29 Greenovation Biotech Gmbh Utilisation of constructs comprising recombination sequence motifs for enhancing gene expression in moss
WO2014147235A1 (fr) 2013-03-22 2014-09-25 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Trousses comprenant des vecteurs viraux à arn à simple brin de sens plus et procédés de production de polypeptides à l'aide des trousses
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US9822376B2 (en) 2002-07-19 2017-11-21 University Of South Carolina Compositions and methods for the modulation of gene expression in plants
EP3260542A1 (fr) 2016-06-20 2017-12-27 Algentech Production de protéine dans des cellules végétales
EP3470420A1 (fr) 2013-08-14 2019-04-17 Institute Of Genetics And Developmental Biology Procédé de modulation de la taille des graines et des organes dans des plantes
WO2019185129A1 (fr) 2018-03-27 2019-10-03 The University Court Of The University Of Glasgow Résistance dérivée d'un pathogène bactérien chez les plantes
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