WO1992014828A1 - Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones - Google Patents

Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones Download PDF

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Publication number
WO1992014828A1
WO1992014828A1 PCT/SE1991/000102 SE9100102W WO9214828A1 WO 1992014828 A1 WO1992014828 A1 WO 1992014828A1 SE 9100102 W SE9100102 W SE 9100102W WO 9214828 A1 WO9214828 A1 WO 9214828A1
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WIPO (PCT)
Prior art keywords
plants
barley
plant
bacterium
transformed
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PCT/SE1991/000102
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English (en)
Inventor
Anneli Tallberg
Eva THÖRN
Richard Walden
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Svalöf Ab
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.)
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Publication date
Application filed by Svalöf Ab filed Critical Svalöf Ab
Priority to PCT/SE1991/000102 priority Critical patent/WO1992014828A1/fr
Publication of WO1992014828A1 publication Critical patent/WO1992014828A1/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

  • the present invention relates to a method for genet transformation of tissues from monocotyledonous plants.
  • the invention also comprises tissues from monocotyledons transformed by means of the method according to the inve tion.
  • Transformed plants are formed by inserting, by means of genetic engineering, genetic material into individual cells (protoplasts) or into an intact tissue. Since the Agrobacterium system has considerable limitations when applied to cereals, transformation systems based on direct gene transfer have been developed for cereals (Paszkowski et al 1986). The drawback of these gene transfer methods, e.g. electroporation and microinjection, is the difficulty of the receptor cells to divide and form intact fertile plants. However, by using standard methods for direct gene transfer to protoplasts from embryogenic suspension cul ⁇ tures, it has been possible to regenerate fertile trans- genic rice plants (Potrykus 1990).
  • the invention relates to a method for genetic trans ⁇ formation of tissues from a monocotyledonous plant, in which method genetic material is inserted into a vector i a bacterium, whereupon the bacterium is co-cultivated wit a microspore culture of the plant to be transformed.
  • the method preferably is applied to cereals, espe ⁇ cially to barley.
  • tis- sues are meant seeds, embryos, proembryos, microspores, pollen etc.
  • the invention also comprises genetically transformed tissues from a monocotyledonous plant which has been transformed by insertion of genetic material into a vecto in a bacterium which is subsequently co-cultivated with a microspore culture of the monocotyledonous plant.
  • the inventive method for transforming tissue organs from monocotyledons, especially cereals is based on tech niques for cultivating isolated microspores.
  • microspores cultivate in vitro have the capability of dividing and differentiat ing to embryos.
  • the development has reached a stage when the surrounding wall bursts, and a proembryo is released.
  • the smaller number of cells neede for regeneration the better is the cultivation system suited for gene transfer.
  • a 6-day-old proembryo consists of some hundred cells, it is still very small as compared with a fully developed embryo. A transformation at this stage means good prospects of obtaining embryos which largely consist of transformed tissue.
  • the invention is based on co-cultivation of microspores of the plant to be transformed with bacteria cells which contain a vector having the genetic material which is desired to be inserted in the host plant.
  • a bacterium which has appeared to function well in the field of the invention is Agrobacterium, especially Agrobacterium tume aciens.
  • the transformation process can be divided into four 5 steps, i.e.
  • Fig. 1 shows the vector pPCV 002
  • Fig. 2 shows the vector pCVHPT GUS
  • Fig. 3 shows Southern blot I with the HPT fragment as the probe.
  • Fig. 4 shows Southern blot II 500 bp vector sequence as the probe
  • Fig. 5 shows Southern blot III with the HPT fragment as 25 the probe, and
  • Fig. 6 is a photo of a regenerated fertile transgenic bar ⁇ ley plant. The invention is described in more detail in the Exam ple below which is not limiting. 30 EXAMPLE
  • Isolated microspores from barley are used as starting material for the experiment, since a well functioning 35 method for cultivating isolated barley microspores and for regenerating plants therefrom is available.
  • Agrobac ⁇ terium tumefaciens was selected as a suitable bacterium for insertion of the genetic material.
  • the Agrobacterium tumefaciens used includes the vec ⁇ tor pCVHPT GUS (Fig. 2) which results in hygromycin resis tance.
  • the vector pCVHPT GUS is based on another vector, pPCV 002 (Fig. 1) (Walden et al 1990).
  • pCVHPT GUS a gene for hygromycin resistance was inserted, viz. HPT 2030 bp, at the EcoRI-Sall-site in the polylinker molecule from pPCV 002.
  • the reporter gene for beta glucuronidase (GUS) (Jefferson et al 1986) replaced the NPT II gene in pCVHPT GUS between pNOS and pAccs from pPCV 002.
  • Barley microspores at a late single nucleus to early double nuclei stage are isolated from ear which has been refrigerated as +4°C for 4 weeks.
  • the microspores are cul tivated in 7 cm Petri dishes in the dark at 25°C in N6 medium (Chu 1978) mixed with 1.75 mg/1 2,4-D 0.25 mg/1 kinetin 63 mg/1 maltose.
  • the culture medium is replaced by a new N6 medium mixed with 0.5 % MES
  • the material After adding the bacteria, the material is placed in the dark where it is first agitated at 30-40 rpm for 20 min. and is then allowed to stand for 3 days. Selection and Regeneration of Transgenic Barley Plants After the period of 3 days, the culture medium is replaced by selective N6 medium mixed with 10 mg/1 hygromycin 200 mg/1 Claforan.
  • Claforan The function of Claforan is to stop the growth of the excess of Agrobacterium cells, while the hygromycin is added for selection of transformed tissue only, to which the hygromycin gene has been transferred.
  • the embryo material is transferred to a regeneration medium consist ⁇ ing of J-25-8-medium (Jensen 1983) mixed with 0.5 mg/1 IAA 1 mg/1 kinetin
  • barley plants About 8 weeks after the initial microspore isolation, barley plants have been regenerated. They are transferred to soil and placed in a greenhouse.
  • This consists of a 500 bp vec ⁇ tor sequence from pCVHPT GUS which is not integrated between the T-DNA boundary sequences and which thus canno be transferred to the barley genome.
  • This additional con ⁇ trol is performed in order to determine that the initial positive signals do not derive from surviving Agrobac ⁇ terium cells. The result obtained therefore is a complete ly blank membrane without any signals (Fig. 4).
  • GUS analysis The presence of the GUS gene (the gene for beta glu- curonidase from E. coli) is analysed in nonfreeze-dried plant material. The activity of beta glucuronidase is analysed on leaf extracts from the regenerated barley plants by visual examination. Determination of the chromosome number:
  • Transformed, regenerated barley plants have been obtained from barley microspores treated according to the method described above. A total of 150 barley plants have been regenerated, 10% of which are of albino type.
  • Fig. 6 shows a photo of a regenerated, fertile transgenic plant which has been obtained by the method according to the invention.
  • Fig. 1 pPCV002: LB and RB are the right and left T-DNA boundary; oriV and oriT, replication start functioning in Agrobacterium; ori, bom, ColEl, replication start and mobilising sequence functioning in E.
  • Fig. 2 pCVHPT GUS: LB and RB are the right and left T-DNA boundary; oriV and oriT, replication start functioning in AgroJbacterium; ori, bom, ColEl, replication start and mobilising sequence functioning in E.
  • coli Amp ampicillin resistance gene: pNos, nopaline syntase promotor; GUS, ⁇ -Gucuronidase gene; pAocs, sequence for octopine syntase polyA; HPT, hygromycin resistance gene.
  • pCVHPT GUS is based on pPCV002.
  • Line 1 is DNA from transformed barley plan .
  • Lines 2 and 3 are HPT fragments from pCVHPT GUS.
  • Fig. 4 Regenerated transformed barley plant.

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

Abstract

Procédé de transformation génétique de tissus provenant d'une plante monocotylédone. On introduit la matière génétique dans un vecteur dans une bactérie qui est ensuite cultivée en combinaison avec une culture microspore de la plante qu'on désire transformer. Cette invention concerne également des tissus transformés génétiquement à l'aide du procédé décrit, lesdits tissus provenant d'une plante monocotylédone.
PCT/SE1991/000102 1991-02-14 1991-02-14 Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones WO1992014828A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/SE1991/000102 WO1992014828A1 (fr) 1991-02-14 1991-02-14 Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones

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Application Number Priority Date Filing Date Title
PCT/SE1991/000102 WO1992014828A1 (fr) 1991-02-14 1991-02-14 Procede de transformation genetique d'organes tissulaires provenant de plantes monocotyledones

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WO1992014828A1 true WO1992014828A1 (fr) 1992-09-03

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029419A1 (fr) * 1995-03-17 1996-09-26 Hoechst Schering Agrevo Gmbh Microspores enbryogenes transformees destinees a la production de plantes homozygotes fertiles
WO2001041557A2 (fr) * 1999-12-10 2001-06-14 University Of Guelph Embryogenese et regeneration de plantes a partir de microspores
EP1983056A1 (fr) * 1992-07-07 2008-10-22 Japan Tobacco Inc. Procédé de transformation de monocotylédones
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
WO2010061186A2 (fr) 2008-11-25 2010-06-03 Algentech Sas Procédé de transformation de plastide de plante
WO2010061187A2 (fr) 2008-11-25 2010-06-03 Algentech Sas Procédé de transformation de mitochondries de plante
WO2010084331A2 (fr) 2009-01-26 2010-07-29 Algentech Sas Ciblage de gène dans des plantes
WO2011017288A1 (fr) 2009-08-05 2011-02-10 Chromocell Corporation Plantes, microbes et organismes enrichis
EP2357239A1 (fr) 2009-10-29 2011-08-17 Universität zu Köln Procédés et moyens pour un système marqueur sélectionnable dans les plantes
US8153863B2 (en) 2007-03-23 2012-04-10 New York University Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity
WO2013057208A1 (fr) 2011-10-18 2013-04-25 Targeted Delivery Technologies Limited Compositions et procédés pour la réduction de la prolifération et de la viabilité d'agents microbiens
WO2013093637A2 (fr) 2011-12-19 2013-06-27 Szkola Glowna Gospodarstwa Weijskeigo W Warszawie Procédés de traitements des plantes et moyens associés
WO2015067943A1 (fr) 2013-11-11 2015-05-14 Plant Bioscience Limited Procédés de modulation de la taille des semences et des organes chez les plantes
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

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003776A1 (fr) * 1984-12-21 1986-07-03 Plant Genetic Systems N.V. Procede de preparation de cellules de plantes monocotyledones a transformation genetique stable
WO1988009374A1 (fr) * 1987-05-22 1988-12-01 Max-Planck-Gesellschaft Zur Förderung Der Wissensc Embryons de plantes utiles servant de systeme de captage d'information genetique exogene
GB2211204A (en) * 1987-10-20 1989-06-28 Oji Paper Co Process for production of plant transformant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003776A1 (fr) * 1984-12-21 1986-07-03 Plant Genetic Systems N.V. Procede de preparation de cellules de plantes monocotyledones a transformation genetique stable
WO1988009374A1 (fr) * 1987-05-22 1988-12-01 Max-Planck-Gesellschaft Zur Förderung Der Wissensc Embryons de plantes utiles servant de systeme de captage d'information genetique exogene
GB2211204A (en) * 1987-10-20 1989-06-28 Oji Paper Co Process for production of plant transformant

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 357, Biotechnology abstracts, Dialog accession no. 116315, HESS D et al.: "Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.) - Agrobacterium tumefaciens transformation of wheat to kanamycin-resistance by pollen transfer"; & *
DIALOG INFORMATION SERVICES, File 55, BIOSIS 85-91, Dialog accession no. 7666836, CREISSEN G et al.: "Agrobacterium and microprojectile mediated viral DNA delivery into barley microspore-derived cultures"; & PLANT CELL REP 8 (11), 1990, 680-683. *
DIALOG INFORMATION SERVICES, File 55, BIOSIS 85-91, Dialog accession no. 7731304, DENG W-Y et al.: "Agrobacterium Tumefaciens can transform triticum-aestivum and hordeum-vulgare of gramineae"; & SCI CHINA SER B CHEM LIFE SCI EARTH SCI 33 (1), 1990, 27-34. *
PROC. NATL. ACAD. SCI., Vol. 87, June 1990, ERIC MESSENS et al.: "A nontransformable Triticum monococcum monocotyledonous culture produces the potent Agrobacterium vir-inducing compound ethyl ferulate", see page 4368 - page 4372. *
TIBTECH, Vol. 7, October 1989, INGO POTRYKUS: "Gene transfer to cereals: an assessment", see page 269 - page 273. *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1983056A1 (fr) * 1992-07-07 2008-10-22 Japan Tobacco Inc. Procédé de transformation de monocotylédones
EP0737748A1 (fr) * 1995-03-17 1996-10-16 Hoechst NOR-AM AgrEvo Inc. Production effective de plantes transgéniques fertiles homozygènes à partir de microspores fraîches
AU710201B2 (en) * 1995-03-17 1999-09-16 Agrevo Canada, Inc. Transformed embryogenic microspores for the generation of fertile homozygous plants
US6316694B1 (en) 1995-03-17 2001-11-13 Agrevo Canada, Inc. Transformed embryogenic microspores for the generation of fertile homozygous plants
CN1110562C (zh) * 1995-03-17 2003-06-04 农业发展加拿大公司 用于产生可育纯合植株的转化的胚发生小孢子
WO1996029419A1 (fr) * 1995-03-17 1996-09-26 Hoechst Schering Agrevo Gmbh Microspores enbryogenes transformees destinees a la production de plantes homozygotes fertiles
WO2001041557A2 (fr) * 1999-12-10 2001-06-14 University Of Guelph Embryogenese et regeneration de plantes a partir de microspores
WO2001041557A3 (fr) * 1999-12-10 2001-11-29 Univ Guelph Embryogenese et regeneration de plantes a partir de microspores
US6812028B1 (en) 1999-12-10 2004-11-02 University Of Guelph Embryogenesis and plant regeneration from microspores
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
US8153863B2 (en) 2007-03-23 2012-04-10 New York University Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity
US9464296B2 (en) 2007-03-23 2016-10-11 New York University Methods of affecting nitrogen assimilation in plants
EP2471934A2 (fr) 2007-05-22 2012-07-04 Plant Bioscience Limited Composition et procédé de modulation du dévelopment du chevelu racinaire de plantes
WO2008142364A2 (fr) 2007-05-22 2008-11-27 Plant Bioscience Limited Composition et methode de modulation du developpement du chevelu racinaire de plantes
EP2471932A2 (fr) 2007-05-22 2012-07-04 Plant Bioscience Limited Composition et procédé de modulation du dévelopment du chevelu racinaire de plantes
EP2471933A2 (fr) 2007-05-22 2012-07-04 Plant Bioscience Limited Composition et procédé de modulation du dévelopment du chevelu racinaire de plantes
EP2471931A2 (fr) 2007-05-22 2012-07-04 Plant Bioscience Limited Composition et procédé de modulation du dévelopment du chevelu racinaire de plantes
EP2471935A2 (fr) 2007-05-22 2012-07-04 Plant Bioscience Limited Composition et procédé de modulation du dévelopment du chevelu racinaire de plantes
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
WO2011017288A1 (fr) 2009-08-05 2011-02-10 Chromocell Corporation Plantes, microbes et organismes enrichis
EP2357239A1 (fr) 2009-10-29 2011-08-17 Universität zu Köln Procédés et moyens pour un système marqueur sélectionnable dans les plantes
WO2013057208A1 (fr) 2011-10-18 2013-04-25 Targeted Delivery Technologies Limited Compositions et procédés pour la réduction de la prolifération et de la viabilité d'agents microbiens
WO2013093637A2 (fr) 2011-12-19 2013-06-27 Szkola Glowna Gospodarstwa Weijskeigo W Warszawie Procédés de traitements des plantes et moyens associés
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
WO2015067943A1 (fr) 2013-11-11 2015-05-14 Plant Bioscience Limited Procédés de modulation de la taille des semences et des organes chez les plantes
EP4269600A2 (fr) 2013-11-11 2023-11-01 Plant Bioscience Limited Procédés de modulation de la taille des semences et des organes chez les plantes
EP3260542A1 (fr) 2016-06-20 2017-12-27 Algentech Production de protéine dans des cellules végétales
WO2017220539A1 (fr) 2016-06-20 2017-12-28 Algentech Production de protéines dans des cellules végétales

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