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
• • 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
  • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• • 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

Definitions

• the method according to the invention is a useful alternative to previously known introduction methods.
• the method provides a new approach involving only moderate costs, said method being carried out quickly and simply. Based on introduction experiments already conducted it can be predicted that the method will also turn out to be superior when used for a great number of other biological materials, where known methods are insufficient.
• the plant cells are subjected to an ultrasound treatment considerably milder than the ultrasound treatment used for homogenizing or lysing cells. It is important that the treatment is suitably mild so that a sufficient number of plant cells remain viable.
• the plant cell suspension can advantageously be exposed to ultrasonic waves of a frequency range of from 5 kHz to 10 MHz, particularly from 10 to 100 kHz, and an electric output power (i.e.
• the method according to the invention has advantageously been used to introduce molecules into cells of sugar beet and tobacco plants.
• the medium can be any suitable, conventional medium for cells or protoplasts.
• the method according to the invention uses a technique presumbably resulting in a temporary moderate weakening of both the cell membrane and the cell wall, which is shown in intact plant cells in the following examples. The examples thus show that plasmid DNA can penetrate plant cells having been treated with ultrasound.
• CPW is an aqueous solution of a mixture of inorganic salts comprising i.a. approx. 10 mM Ca , described by Frearson et al., (Dev. Biol., 33, 130-137, 1973).
• a cell suspension for ultrasound treatment is prepared by removing cells 3 to 4 days after sub-culturing and washing them twice with CPW 13S (i.e. CPW containing 13% sorbitol), finally suspending said cells in CPW 13S at a ratio of 1 part by volume cells to 4 parts by volume CPW 13S. Then plasmid DNA is added to the suspension in 0.35 ml CPW containing 21% sucrose and plant cells (500.000/ml) in an Eppendorf tube, the final plasmid concentration being 45 ⁇ g/ml.
• CPW 13S i.e. CPW containing 13% sorbitol
• the plasmid DNA used is a plasmid coding for the marker enzyme chloramphenicol-acetyltransferase (CAT) , in this case the plasmid pCaMVCN having the code 27-4909, available from Pharmacia LKB Biotechnology, Uppsala, Sweden.
• CAT chloramphenicol-acetyltransferase
• the present example illustrates the introduction of plasmid DNA into intact sugar beet cells of the genotype Ml.
• the suspension culture of sugar beet cells is maintained by sub-culturing as described above and is treated with ultrasound, cultured and analysed in the manner described.
• the results of the measured CAT-activity appear from Table 1.
• the present example illustrates the introduction of plasmid DNA into intact tobacco cells.
• the cells are removed 3 to 4 days after sub-culturing and washed twice with CPW 13S (i.e. CPW containing 13% sorbitol), finally suspending said cells in CPW 13S at a ratio of 1 part by volume cells to 4 parts by volume CPW 13S.
• Samples of 0.35 ml each are taken out and the plasmid pCaMVCN is added to each sample, the final plasmid concentration being 100 ⁇ g/ml.
• the cells are then subjected to ultrasound treatment under the conditions appearing from Table 2. After culturing for 2 days in the above-mentioned medium the cells are extracted and their CAT-activity is measured. The results appear from Table 2.
• the method according to the invention can be used to introduce molecules into intact plant cells.

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

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• 1989
  • 1989-06-29 DK DK325189A patent/DK168302B1/da not_active IP Right Cessation
• 1990
  • 1990-06-28 WO PCT/DK1990/000166 patent/WO1991000358A1/en not_active Application Discontinuation
  • 1990-06-28 JP JP51026590A patent/JPH05500304A/ja active Pending
  • 1990-06-28 AU AU60432/90A patent/AU645260B2/en not_active Expired - Fee Related
  • 1990-06-28 EP EP19900910596 patent/EP0480971A1/en not_active Ceased

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