GB2200367A - Method of preparing genetically transformed plant matter - Google Patents

Method of preparing genetically transformed plant matter Download PDF

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Publication number
GB2200367A
GB2200367A GB08702006A GB8702006A GB2200367A GB 2200367 A GB2200367 A GB 2200367A GB 08702006 A GB08702006 A GB 08702006A GB 8702006 A GB8702006 A GB 8702006A GB 2200367 A GB2200367 A GB 2200367A
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GB
United Kingdom
Prior art keywords
cell
plant matter
plants
transformed plant
genetically transformed
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.)
Granted
Application number
GB08702006A
Other versions
GB2200367B (en
GB8702006D0 (en
Inventor
Pavel Viktorovich Melnikov
Taras Petrovich Pasternak
Jury Jurievich Gleba
Konstantin Merkurievich Sytnik
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.)
INST BOTAN IM N G KHOLODNOGO A
Original Assignee
INST BOTAN IM N G KHOLODNOGO A
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Publication date
Application filed by INST BOTAN IM N G KHOLODNOGO A filed Critical INST BOTAN IM N G KHOLODNOGO A
Priority to CH316/87A priority Critical patent/CH670655A5/de
Priority to GB8702006A priority patent/GB2200367B/en
Priority to JP62026500A priority patent/JPS63196270A/en
Publication of GB8702006D0 publication Critical patent/GB8702006D0/en
Priority to DE19873738874 priority patent/DE3738874A1/en
Publication of GB2200367A publication Critical patent/GB2200367A/en
Application granted granted Critical
Publication of GB2200367B publication Critical patent/GB2200367B/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection

Abstract

A method of preparing genetically transformed plant matter comprises transformation of plant matter by microinjection of a DNA molecule or an autonomously replicating cell organelle into a protoplast, a single cell, or a cell in the structure, with subsequent selection of cell lines and plants with novel hereditary properties. The proposed method finds application for selecting novel plant species, including argricultural plants, characterized by definite properties. The microinjections may be performed under pressure or under the effect of an electric field.

Description

METHOD OF PREPARING GENETICALLY TRANSFORMED PLANT MATTER The present invention relstes to genetic engineering of plants and more particularly to a method of preparing genetically transformed plant objects, the method being used for selection of new types of plants, including agricultural ones, characterized by definite properties such as specific resistance to herbicides, specific resistance to pathogenic viruses and microorganisms, a modified amino acid composition, an enhanced useful biomass content, an increased stability towards unfavourable environmental factors, and others.
In the herein-proposed method of preparing genetically transformed plant objects by way of introduction of a transforming factor into an object-recipient with subsequent selection of cell lines and plants with novel hereditary promerties, according to the invention, as a transforming factor use is made of a D.TA molecule or an autonomously rerlicating cell organelle and a protoplast, single cell, or a cell in the structure are used as an object-recipient, the transforming factor being introduced into the object-recipient by m.icro- injection. icroinjections can be accomplished with the help of any suitable method.It is reasonable to carry out microinjection under pressure or under the action of an electric field, The herein-crogosed method enhances the transformation efficiency to AOC,? , which exceeds 10-20 times the transformation efficiency of the priorer methods, whereas the consumption of the transforming factor is considerably decreased.
The herein-proposed method makes it possible to use a protoplast, a single cell, and the cell in the structure as objects-recipients; DNA molecules and autonomously replicating cell organelles are used as the transforming factor.
This increases the number of species involved into genetic -engineering manipulations, including such important agri- cultural plants as, for example, cereals.
The herein-!rroDosed method is accomplished in the following way. A protoplast, a single cell, or a cell in the structure (microcallus, embryogenic callus, embryoid), used as an object-recipient, in a corresponding nutrient solution are placed into a microchamber mounted on a microsco?e stage and fixed with the help of, for instance, a sucker, by nouring into agar, mechanically. A glass micropipette filled with a transforming factor (plasmid, cosmid, chloroplast) in a corresponding medium is introduced into said object -recipient with the aid of a micromanipulator, the introduction being performed under visual microscopic control.
Microinåection is accomplished under pneumatic or hydraulic pressure or under the effect of an electric field. The volume of the introduced solution varies from 1 to 10 ico- litres. All operations are carried out under sterile conditions. The injected objects-reciients are transferred onto a selective nutrient medium, the choice thereof being determined by the transforming factor. The choice of transformants and accumulation of their biomass are performed upon repeated transfer of objects-recipients on selective media. Plants are regenerated on the selective media from the tranformed cell lines and cell structures.
or a better understanding of the present invention specific examples of realizing the proposed method are given hereinbelow by way of illustration.
Example 1 A micropipette with an end diameter of about 1 is introduced into a protoplast isolated from Nicotiana debnsyi callus and 1-2 picoliters of a buffer solution (10 mid tris, pH 7.2, 1 mM sodium ethylenediaminotetraacetate, 5 it KC1) containing pSV2NeO plasmid in an amount of about 100 replicas per picoliter of buffer are pressed out with the aid of a hydraulic system. In the course of microinjection up to initial divisions protoplast is in a Caboche nutrient medium containing 0.1 wit.% of agarose. icrocolonis formed from the injected protoplasts are transferred onto Camborg medium containing 60 mg/dm3 of the antibiotic kanamycin sulphate.Eight cell lines were obtained from 53 injected pro- toplasts, said cell lines being able to grow on a medium containing up to 120 mg/dm3 of the antibiotic. The transformation efficiency was 15. From three resistant lines obtained on the Gamburg medium without addition of phytohormones plants were regenerated able to grow and take root on the madium with kanamycin.
Example 2 The procedure is similar to that described in Example 1.
Single cells of Nicotiana tabacum are used as an object-reci pient and pIGV23Neo plasmid is used as a transforming agent.
Microiniection is performed with the aid of a pneumatic system. From 20 injected cells three cell lines were selected able to grow on the Gamburg medium containing up to 120 mg/dm3 of kanamycin sulphate.
Example 3 The procedure is similar to that described in Example 1.
As an object-recipient use is made of cells in microcallus Lycopersicon esculentum (from 8 to 30 cells) in a Satin medium containing 0.3 wt.% of agarose. Plasmid pGV0319 was used as a transforming sgent. In accord with the properties coded by said plasmid, the selection of transformants is performed on a hormone-free Sachin medium. Introduction of plasmid into 95 cells yielded 19 cell lines able to grow on a hormone-free medium. The transformation efficiency was 19.
Example 4 The procedure is similar to that descibed in Example 1.
Single cells of Nicotiana tabacum were used as an object-recipient and cosmid pGA 471 was used as a transforming anent.
injection was performed under the action of a 5-10 V electric field by several 0.5 pulses with the current intensity of up to 0.1 microampere, Prom 23 cells transformed as described above 6 cell lines were obtained able to ,row on a medium containing 120 mg/dm3 of kanamycin sulphate. The transformation efficiency was about 2591. ?rom two cell lines plants were regenerated able to grow and take roots on R medium containing kanamycin.
Example 5 The procedure is similar to that described in Example 4.
As a recipient use is made of cells of embryogenic callus of Triticum aestivum wheat, idironovskaya rannyaja, said cells being fixed mechanically. rom 20 to 100 cells were - injected to each of 8 calluses. Said calluses were transferred to Murashige-Skoog medium containing 2 mg/dm3 of 2,4-dichlorophenoxyacetic acid and 60 mg,'dm3 of kanamycin sulphate. Six cell lines were obtained able to grow on a medium with kanamycin sulphate.
Example 6 The procedure is performed as described in Example 1.
Single white cells of plastomic mutant of Ticotiana tabacum of the IP line were used as an object-recipient and chioroplasts isolated from leaves of Nicotiana tabacum of the SR2 line were used as autonomously replicating organelles. Said chloroplasts were characterized by the resistance to the antibiotic streptomycin, the resistance being coded b the clloroplasts. A micropipette with the end diameter to 8 sum was filled with suspension of chloroplasts in Jensen-3asshem medium and 5-10 chloroplasts were pressed out into a cell.
After initial divisions the injected cells were transferred onto the Gamburg medium containing 1 mgXcm3 streptomycin sulphate. From 16 thus injected cells 3 cell lines ere selected, said lines being characterized by green colour and the ability to growth on a medium containing 1 mg/cm of the antibiotic.

Claims (3)

CLAIMS:
1. A method of preparing genetically transformed plant matter in which the transformation is performed by microinjection of a DNA molecule or an autonomously replicating cell organelle into a protoplast, a single cell, or a cell in the structure with subsequent selection of cell lines and plants with novel hereditary properties.
2. A method as claimed in claim 1, wherein said microinjections are performed under pressure or under the effect of an electric field.
3. A method as claimed in the above claims, substantially as described herein with reference to Examples 1 to 6.
GB8702006A 1987-01-29 1987-01-29 Method of preparing genetically transformed cellular plant matter by micro- injection. Expired - Fee Related GB2200367B (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CH316/87A CH670655A5 (en) 1987-01-29 1987-01-29
GB8702006A GB2200367B (en) 1987-01-29 1987-01-29 Method of preparing genetically transformed cellular plant matter by micro- injection.
JP62026500A JPS63196270A (en) 1987-01-29 1987-02-09 Production of genetically transformed plant
DE19873738874 DE3738874A1 (en) 1987-01-29 1987-11-16 Process for the preparation of genetically transformed plant objects

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE3702659 1987-01-29
GB8702006A GB2200367B (en) 1987-01-29 1987-01-29 Method of preparing genetically transformed cellular plant matter by micro- injection.
DE19873738874 DE3738874A1 (en) 1987-01-29 1987-11-16 Process for the preparation of genetically transformed plant objects

Publications (3)

Publication Number Publication Date
GB8702006D0 GB8702006D0 (en) 1987-03-04
GB2200367A true GB2200367A (en) 1988-08-03
GB2200367B GB2200367B (en) 1990-08-08

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Country Status (3)

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CH (1) CH670655A5 (en)
DE (1) DE3738874A1 (en)
GB (1) GB2200367B (en)

Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO1999036563A1 (en) * 1998-01-14 1999-07-22 Emed Corporation Electrically mediated cellular expression

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EP0331083A3 (en) * 1988-03-02 1990-11-28 Schweizerische Eidgenossenschaft Eidgenössische Technische Hochschule (Eth) Method for the production of transgenic plants
FI902635A0 (en) * 1989-05-29 1990-05-25 Eidgenoess Tech Hochschule FOERFARANDE FOER FRAMSTAELLNING AV TRANSGENA VAEXTER.
US5550318A (en) * 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US6803499B1 (en) 1989-08-09 2004-10-12 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
WO1991002071A2 (en) * 1989-08-09 1991-02-21 Dekalb Plant Genetics Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US7705215B1 (en) 1990-04-17 2010-04-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US6946587B1 (en) 1990-01-22 2005-09-20 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5484956A (en) * 1990-01-22 1996-01-16 Dekalb Genetics Corporation Fertile transgenic Zea mays plant comprising heterologous DNA encoding Bacillus thuringiensis endotoxin
JP3209744B2 (en) 1990-01-22 2001-09-17 デカルブ・ジェネティクス・コーポレーション Transgenic corn with fruiting ability
US6329574B1 (en) 1990-01-22 2001-12-11 Dekalb Genetics Corporation High lysine fertile transgenic corn plants
US6777589B1 (en) 1990-01-22 2004-08-17 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
DE4013099A1 (en) * 1990-04-25 1991-10-31 Hoechst Ag Transforming immature somatic plant, esp. maize, embryos - by treating, in dry state, with nucleic acid soln., esp. for introducing resistance to phosphinothricin
US6395966B1 (en) 1990-08-09 2002-05-28 Dekalb Genetics Corp. Fertile transgenic maize plants containing a gene encoding the pat protein
ATE191931T1 (en) * 1990-11-23 2000-05-15 Plant Genetic Systems Nv METHOD FOR TRANSFORMING MONOCOTYL PLANTS
US5405765A (en) * 1991-08-23 1995-04-11 University Of Florida Method for the production of transgenic wheat plants
CA2104341A1 (en) * 1992-08-19 1994-02-20 Charles L. Armstrong Method for transforming monocotyledonous plants
US6118047A (en) 1993-08-25 2000-09-12 Dekalb Genetic Corporation Anthranilate synthase gene and method of use thereof for conferring tryptophan overproduction
US5780709A (en) * 1993-08-25 1998-07-14 Dekalb Genetics Corporation Transgenic maize with increased mannitol content

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EP0175966A1 (en) * 1984-09-25 1986-04-02 Calgene, Inc. Plant cell microinjection technique

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036563A1 (en) * 1998-01-14 1999-07-22 Emed Corporation Electrically mediated cellular expression

Also Published As

Publication number Publication date
GB2200367B (en) 1990-08-08
GB8702006D0 (en) 1987-03-04
DE3738874C2 (en) 1991-06-27
CH670655A5 (en) 1989-06-30
DE3738874A1 (en) 1988-11-17

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