WO1986003776A1 - Procede de preparation de cellules de plantes monocotyledones a transformation genetique stable - Google Patents

Procede de preparation de cellules de plantes monocotyledones a transformation genetique stable Download PDF

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Publication number
WO1986003776A1
WO1986003776A1 PCT/EP1985/000726 EP8500726W WO8603776A1 WO 1986003776 A1 WO1986003776 A1 WO 1986003776A1 EP 8500726 W EP8500726 W EP 8500726W WO 8603776 A1 WO8603776 A1 WO 8603776A1
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Prior art keywords
plant cells
plasmid
process according
dna
hybrid
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PCT/EP1985/000726
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English (en)
Inventor
Jean Pierre E. C. Hernalsteens
Marc Charles Van Montagu
Josef S. Schell
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Plant Genetic Systems N.V.
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Application filed by Plant Genetic Systems N.V. filed Critical Plant Genetic Systems N.V.
Publication of WO1986003776A1 publication Critical patent/WO1986003776A1/fr

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

Definitions

  • Ti-piasmids still contain the border sequences and consequently transfer T-DNA without tumor induction.
  • An example of such a Ti-plasmid derived vector for plant engineering is pGV 3850.
  • This Ti-derivative has proven very useful. It contains a substitution of the internal T-DNA genes by the commonly used cloning vehicle pBR322. Plant cells transformed by pGV 3850 have the same regenerative capacity as untransformed cells and several procedures have been developped to obtain intact plants which contain the pGV 3850 T-DNA.
  • pGV 3850, through the pBR 322 sequences present in its T-DNA, is an efficient acceptor plasmid for gene transfer experiments in plant cells (see European patent application No. 0 116 718).
  • genes cloned in pBR 322-like plasmids are transfered to Agrobacterium and inserted via homologous recombination into the pGV 3850 T-DNA in a single experimental step.
  • Another major advance in the application of T-DNA as a vector is the use of plant regulatory sequences to express chimeric genes in plants.
  • These chimeric genes contain a promotor region derived from a gene which is naturally expressed in plant cells, the sequence to be expressed, and preferentially a 3'-non-translated region containing a poly-adenylation site of a gene which is naturally expressed in plant cells.
  • a promotor region derived from a gene which is naturally expressed in plant cells
  • the sequence to be expressed and preferentially a 3'-non-translated region containing a poly-adenylation site of a gene which is naturally expressed in plant cells.
  • dominant selectable markers for plant cells have been constructed.
  • the genes involved in the synthesis of opines are encoded by the T-DNA of Agrobacterium, but the regulatory sequences involved in their expression are of an eucaryotic type. This explains why these genes are expressed in a plant environment.
  • the observation of the presence of opines in the wound tissue of infected monocotyledonous plants suggested at least a temporarily transfer and expression of Ti-plasmid genes into the monocotyledonous plant.
  • these data did not provide any prove for stable transfer of Ti- plasmid DNA into the plant cell. The investigators did not show the monocot tissue to produce opines after sub-culture under aseptical conditions.
  • the object of the invention is to provide a process for preparing transformed monocotyledonous plant cells which are genetically stable.
  • a further object of the invention is the preparation of genetically transformed monocotyledonous plant cells as well as plants regenerated therefrom.
  • the present invention relates to a process for preparing transformed plant cells which are genetically stable comprising the transformation of plant cells using Agrobacterium harbouring a hybrid Ti-plasmid vector, characterized in that the plant cells are derived from a monocotyledonous plant.
  • the invention makes use of hybrid Ti-plasmids such as those described in European Patent Application No. 0116718. These are Ti-plasmids, from which the oncogenic genes or tumor inducing genes have been removed from the T-DNA.
  • Ti-plasmids can be used which contain a gene of interest operatively linked to a suitable oromoter, e.g. the nopaline-synthase-promoter. Such a combination is also called a chimeric gene.
  • these plasmids contain chimeric antibiotic resistance genes, which provide a dominant selectable marker for plant cells.
  • Fig. 1 Construction of the intermediate plasmid vector pLGVneo1103. a
  • Step 1 Construction of/290 bp Sau 3A fragment containing the nopaline synthase promoter.
  • the construction is similar to the one described bv Herrera- Estrella et al. (ref. 2 ) .
  • the starting plasmid is PLGV13 (ref.2). 20 ⁇ g of pLGV13
  • T4 DNA ligase (Biolabs, New England) with 2 units T4 DNA ligase in a total volume of 20 ⁇ l. After the T4 ligase was inactivated at 68° C for 10 minutes, each ligation mix was digested with 20 units BglII for 1 h at 37° C. Subsequently,
  • DNA 50 ng DNA were recircularized with 0.1 unit T4/ligase in a total volume of 100 ⁇ l for 20 h at 4° C.
  • T4/ligase 0.1 unit T4/ligase in a total volume of 100 ⁇ l for 20 h at 4° C.
  • One fifth of this ligation mixture was transformed into competent
  • E. coli K 514 cells (ref. 3 ) as described by Dagert and
  • deletion end points in the plasmids were first analyzed by measuring the size of the newly generated Bcll-Bglll fragments of the recombinant plasmids. The nucleotide sequence of the exact deletion end points in some plasmids were determined.
  • the nos promoter can be isolated as a
  • Step 2 Insertion of Pnos in front of the neo gene.
  • the 290 bp Bcll-Bglll fragment containing the nopaline synthase promoter (Pnos) was purified from a 2 % agarose gel by electroelution and inserted into pKM109-2 (ref. 7 ) .
  • One of the resulting recombinants, pLGVneo011 contains the Pnos fragment in the orientation such that it will direct transcription of the neomycin phosphotransferase gene (neo) in plant cells.
  • the orientation was determined by SacII-EcoRI digestion.
  • Step 3 Insertion of the 3' end of the octopine synthase gene behind the neo gene.
  • a fragment containing the 3' untranslated region of the octopine synthase gene was isolated from pGV99 (ref. 8 )) . This was achieved by digesting 20 ⁇ g of pGV99 DNA with 20 units of PvuII for 1 h at 37° C, separation of the fragments on a 0.8 % agarose gel and isolation of the 706 bp PvuII fragment by electroelution. The 706 bp PvuII fragment was inserted into the Smal site of pLGVneo011. One of the resulting recombinant plasmids, pLGVneo11 contains the PvuII fragment in the correct orientation with respect to the neo gene.
  • pLGVneo1103 contains a chimeric neo gene consisting of the nopaline synthase promoter, the coding region of the neo gene and the 3' end of the octopine synthase gene, including the polyadenylation signal. The advantage of this chimeric neo gene over the one previously described (ref.
  • the AUG initiation codon for the neomycin phosphotransferase is the first AUG codon in the 5' end of the transcript. This allows optimal translation to occur, and hence to provide resistance levels in plants towards Kanamycin, which are substantially higher
  • Fig. 2 Schematic representation of the T region of PGV3850 and of intermediate vector PLGV1103. Tne crossed lines indicate the regions which were involved in co-integration of PGV3850 with pLGVneo1103. The T region of hybrid Ti-plasmid pGV2302 is represented.
  • Ap,KM gene encoding resp. ampicillin and kanamycin resistance
  • Fig. 3 Detection of nopaline in tissue extracts
  • Example 1 Isolation of a cell culture line which is derived from a monocotyledonous plant and that contains and expresses T-DNA genes of Agrobacterium tumefaciens.
  • Plant cells of Asparagus officinalis L.cv. (Ruhm v. Braunschweig) transformed with an oncogenic Ti-plasmid (e.g. C58) were prepared.
  • the procedure involves the following steps:
  • steps 1 to 10 Using this procedure (steps 1 to 10), a tissue culture line which grows vigorously on hormone-free media was obtained. Testing for nopaline in this tissue as described (steps 11 - 16) shows that it indeed produces significant levels of nopaline (see Fig. 3). It was concluded that T-DNA promoters are active in these cells, with an efficiency comparable to that found in dicots . Genes for hormone independent growth are also expressed since the transformed cells proliferate on hormone free culture medium. The presence of T-DNA in the transformed cell line was further demonstrated by Southern blot analysis of total DNA established from the cell cultures, using cloned T-DNA fragments as radioactive probes. The procedure involves the following steps:
  • Plant cells of Asparagus officinalis L. cv. "Ruhm v. Braunschweig” transformed with a non-oncogenic Ti-plasmid derivative (eg. pGV2302) were prepared.
  • the hybrid Ti-plasmid used in this experiment and carrying a gene of interest was pGV2302.
  • This plasmid is a derivative of pGV3850 which is described in European Patent Application No. 0116718 and was obtained as follows. According to the method of Van Haute et al. (ref. 11) helper plasmids were transferred from GJ23 cells to E. coli K514r-m + containing pGLVneo1103 (for detailed description see below).
  • pGLVneo1103 can also be transferred to the helper plasmid containing GJ23 cells.
  • pGLVneo1103 is mobilized during conjugation from the bacteria strain containing the helper plasmids as well as pGLVneo 1103 to Agrobacterium tumefaciens C 58 C 1 Rif R pGV3850 which carries the hybrid Ti-plasmid pGV3850 (ref. 18). This plasmid transfer is followed by an in vivo recombination event in the recipient strain of Agrobacterium tumefaciens.
  • the result is the recombinant plasmid pGV2302 which carries the genetic information for rifampycin and kanamycin resistance (see Fig. 2) .
  • the strain C 58 C 1 Rif R pGV2302 of Agrobacterium tumefaciens carrying plasmid pGV2302 and the strain of E. coli carrying the plasmid pGLVneo1103 were deposited with the Deutsche Sammlung fiir Mikroorganismen, Göttingen, West Germany, under the accession numbers DSM 3168 and DSM 3169, respectively.
  • pLGVneo1103 (a detailed description of the construction is given in Fig. 1) is an intermediate vector which contains a chimeric gene consisting of the promoter of the nopaline synthase gene (Depicker et al., ref. 14 ), the coding region of the neomycin phosphotransferase gene of Tn5, and the 3' terminal sequences of the octopine synthase gene (Dhaese et al., ref. 1 5 ).
  • the procedure involves the following steps:
  • the calli are about 1 cm in diameter. Devide the calli in pieces of approx. 5 mm diameter and transfer them in 5 cm petridishes containing LS medium with 1 mg/1 BAP, 1 mg/1 NAA, 200 mg/1 glutamine, 250 mg/1 cefotaximum, and 50 mg/1 kanamycin sulfate (Sigma). Use one dish per original callus. Kanamycin sulfate was prepared as a stock solution at 50 mg/ml in distilled water and the pH was adjusted to 5,7 with HCl. The solution was filter-sterilized. The pH of the medium at the moment of its use was 5.7.
  • steps 1 - 11 2 independent tissue culture lines which grow vigorously on media containing 50 mg/1 kanamycin sulfate were obtained. Testing for nopaline in these tissues as described (steps 12 - 17) indicated that both of them indeed produce significant levels of nopaline (see Fig. 3).
  • kanamycin resistant and nopaline containing plants were regenerated from two of the transformed asparagus plants and transferred to the greenhouse.

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Abstract

Procédé de préparation de cellules de plantes monocotylédones à transformation génétique stable, consistant à transformer les cellules monocotylédones en utilisant un Agrobactérium abritant un vecteur de Ti-plasmide hybride.
PCT/EP1985/000726 1984-12-21 1985-12-20 Procede de preparation de cellules de plantes monocotyledones a transformation genetique stable WO1986003776A1 (fr)

Applications Claiming Priority (2)

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EP84116036 1984-12-21
AT84116036.9(EP) 1984-12-21

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0267159A2 (fr) * 1986-11-07 1988-05-11 Ciba-Geigy Ag Procédé de modification génétique de monocotylédones
WO1992014828A1 (fr) * 1991-02-14 1992-09-03 Svalöf Ab Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones
US5595733A (en) * 1987-05-20 1997-01-21 Ciba-Geigy Corporation Methods for protecting ZEA mays plants against pest damage
US6040498A (en) * 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
WO2000058484A2 (fr) * 1999-03-26 2000-10-05 University Of Guelph Transformation de plantes monocotyledones a l'aide d'agrobacterium
US6664109B2 (en) 1985-01-17 2003-12-16 Calgene Llc Transformation system with Ti or Ri plasmid
US7060876B2 (en) 1992-07-07 2006-06-13 Japan Tobacco Inc. Method for transforming monocotyledons
US7161064B2 (en) 1997-08-12 2007-01-09 North Carolina State University Method for producing stably transformed duckweed using microprojectile bombardment
WO2014153032A1 (fr) 2013-03-14 2014-09-25 Evolutionary Genomics, Inc. Identification et utilisation de gènes de la tomate contrôlant la tolérance au sel/à la sécheresse et le goût sucré du fruit
EP2927323A2 (fr) 2011-04-11 2015-10-07 Targeted Growth, Inc. Identification et utilisation de mutants kpr dans des plantes
EP3552482A1 (fr) 2013-10-29 2019-10-16 Biotech Institute, LLC Sélection, production, traitement et utilisation de cannabis spécial
US11530419B2 (en) 2020-10-30 2022-12-20 Fortiphyte, Inc. Pathogen resistance in plants
WO2024052856A1 (fr) 2022-09-09 2024-03-14 Friedrich Alexander Universität Erlangen-Nürnberg Éléments régulateurs chez des plantes et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983001176A1 (fr) * 1981-10-01 1983-04-14 Int Plant Research Inst Procede de modification genetique de cereales avec des vecteurs de transformation
EP0159418A1 (fr) * 1984-04-03 1985-10-30 Rijksuniversiteit Leiden Procédé pour l'incorporation D'ADN étranger dans le génome de plantes monocotylédones

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983001176A1 (fr) * 1981-10-01 1983-04-14 Int Plant Research Inst Procede de modification genetique de cereales avec des vecteurs de transformation
EP0159418A1 (fr) * 1984-04-03 1985-10-30 Rijksuniversiteit Leiden Procédé pour l'incorporation D'ADN étranger dans le génome de plantes monocotylédones

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Nature, Volume 311, 25 October 1984, No. 5988, Reading, Berks, (GB) G.M.S. HOOYKAAS-Van Slogteren et al.: "Expression of Ti Plasmid Genes in Monocotyledonous Plants Infected with Agrobacterium Tumefaciens", pages 763-764, see the whole document (cited in the application) *
Nature, Volume 311, October 1984, No. 5988, Reading, Berks (GB) M.H. DRIMMOND: "Plant Engineering: More Prospects fo Ti Plasmids", page 706, see the whole article *
Science, Volume 222, 18 November 1983, A. CAPLAN et al.: "Introduction of Genetic Material into Plant Cells", pages 815-821, see page 821, column 3, paragrah *
The EMBO Journal, Volume 2, No. 12, 1983, P. ZAMBRYSKI et al.: "Ti Plasmid Vector for the Introduction of DNA into Plant Cells Without Alteration of their Normal Regeneration Capacity", pages 2143-2150, *
The EMBO Journal, Volume 3, No. 12, 1984 J. VELTEN et al.: "Isoltion of a dual Plant Promoter Fragment from the Ti Plasmidof Agrobacterium Tumefaciens", pages 2723-2730, see page 2724, column 1, last paragraph - column 2, paragraph 1; figure 2; page 2726, column 1, paragraph 3;, page 2727, column 2, paragraph 1; page 2728, column 2, lines 27-31; page 2729, column 1, lines 16-33 *
The EMBO Journal, Volume 3, No. 13, 1984, J.P. HERNALSTEENS et al.: "An Agrobacterium Transformed Cell Culture from the Monocot Asparagus Officinalis", pages 3079-3041, see the whole document *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6664109B2 (en) 1985-01-17 2003-12-16 Calgene Llc Transformation system with Ti or Ri plasmid
EP0267159A3 (fr) * 1986-11-07 1990-05-02 Ciba-Geigy Ag Procédé de modification génétique de monocotylédones
EP0267159A2 (fr) * 1986-11-07 1988-05-11 Ciba-Geigy Ag Procédé de modification génétique de monocotylédones
US5595733A (en) * 1987-05-20 1997-01-21 Ciba-Geigy Corporation Methods for protecting ZEA mays plants against pest damage
US5766900A (en) * 1987-05-20 1998-06-16 Novartis Corporation Method of regenerating fertile transgenic Zea mays plants from protoplasts
US5824302A (en) * 1987-05-20 1998-10-20 Novartis Finance Corporation Method of controlling insect larvae comprising feeding an insecticidal amount of a transgenic maize plant expressing a polypeptide having Bt-crystal protein toxic properties
WO1992014828A1 (fr) * 1991-02-14 1992-09-03 Svalöf Ab Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones
US7060876B2 (en) 1992-07-07 2006-06-13 Japan Tobacco Inc. Method for transforming monocotyledons
US7161064B2 (en) 1997-08-12 2007-01-09 North Carolina State University Method for producing stably transformed duckweed using microprojectile bombardment
US6040498A (en) * 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
WO2000058484A3 (fr) * 1999-03-26 2001-01-18 Univ Guelph Transformation de plantes monocotyledones a l'aide d'agrobacterium
WO2000058484A2 (fr) * 1999-03-26 2000-10-05 University Of Guelph Transformation de plantes monocotyledones a l'aide d'agrobacterium
EP2927323A2 (fr) 2011-04-11 2015-10-07 Targeted Growth, Inc. Identification et utilisation de mutants kpr dans des plantes
WO2014153032A1 (fr) 2013-03-14 2014-09-25 Evolutionary Genomics, Inc. Identification et utilisation de gènes de la tomate contrôlant la tolérance au sel/à la sécheresse et le goût sucré du fruit
EP3552482A1 (fr) 2013-10-29 2019-10-16 Biotech Institute, LLC Sélection, production, traitement et utilisation de cannabis spécial
US11530419B2 (en) 2020-10-30 2022-12-20 Fortiphyte, Inc. Pathogen resistance in plants
WO2024052856A1 (fr) 2022-09-09 2024-03-14 Friedrich Alexander Universität Erlangen-Nürnberg Éléments régulateurs chez des plantes et leurs utilisations

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AU5309686A (en) 1986-07-22

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