Claims
- 1. A process for direct gene transfer into plant protoplasts that are amenable to transformation via direct gene transfer, which process consisting:
- isolating plant protoplasts from plant tissue and culturing said protoplasts in a nutrient media customarily used for culturing plant protoplasts;
- pre-incubating said protoplasts at a temperature of from 4.degree. to 10.degree. C. for from 20 minutes to 6 hours in a pre-incubation medium containing Ca.sup.2+, K.sup.+ and Na.sup.+ ions and a suitable carbon source before the actual transformation;
- separating said protoplasts from the pre-incubation medium and resuspending said protoplasts in a transformation medium which contains as an essential component from 6.0 to 60 mM Mg.sup.2+ ions in the presence or absence of Ca.sup.2+ ions;
- directly thereafter, adding a DNA sample to said transformation medium;
- from 0.1 to 10 minutes thereafter, adding polyethylene glycol (PEG) to said transformation medium; and
- incubating said protoplasts and DNA sample in said transformation medium for a period that is sufficient for the incorporation of said DNA into said protoplasts.
- 2. Process according to claim 1, wherein the pH value of the incubation solution is from pH 5.6 to pH 12.
- 3. Process according to claim 2, wherein the pH value of the incubation solution is from pH 7 to pH 10.
- 4. Process according to claim 1, wherein, in addition, neutral DNA without a transforming gene is added in excess as carrier DNA to the transformation solution.
- 5. Process according to claim 4, wherein the said neutral DNA is animal DNA, plant DNA, .lambda.-DNA, plasmid DNA or any DNA suitable for carrying out the process.
- 6. Process according to claim 4, wherein the said neutral DNA is a calf's thymus carrier DNA.
- 7. Process according to claim 4, wherein the concentration of the carrier DNA is from 50 to 70 .mu.g/ml.
- 8. Process according to claim 1, wherein the protoplasts and DNA are jointly incubated in said transformation medium, containing as an essential component from 6.0 to 60 mM Mg.sup.2+ ions in the presence or absence of Ca.sup.2+ ions, in the presence of polyethyleneglycol, for a period of from 10 minutes to 6 hours.
- 9. A process according to claim 1, wherein the protoplasts are from plants that can be regenerated from protoplasts.
- 10. Process according to claim 1, wherein the plant protoplasts used are those from a single species of plant or from a systematic unit subordinate to the species.
- 11. Process according to claim 10, wherein the said protoplasts are obtained from leaves, seedlings, stems, flowers, roots, pollen or embryos.
- 12. Process according to claim 11, wherein the protoplasts are leaf protoplasts.
- 13. Process according to claim 10, wherein the said protoplasts are obtained from cell cultures.
- 14. A process according to claim 1, wherein said DNA sample contains one or more genes under the control of expression signals active in plants which are capable of being expressed and replicated in plant cells and a supporting DNA.
- 15. Process according to claim 14, wherein the concentration of the DNA sample containing one or more genes of any origin under the control of expression signals that are active in plants, and a supporting DNA, is from 2 to 20 .mu.g/ml.
- 16. Process according to claim 15, wherein the concentration of the said DNA sample is from 5 to 10 .mu.g/ml.
- 17. Process according to claim 14, wherein the gene used is a gene of which the structural part is of plant, animal, microbial, viral or synthetic origin and of which the expression signals are of plant, animal or synthetic origin.
- 18. Process according to claim 17, wherein the transforming gene is composed of portions of more than one gene from the same organism.
- 19. Process according to claim 17, wherein the said transforming gene has a structural part that imparts a useful and desirable property to the plant being transformed.
- 20. Process according to claim 19, wherein the said structural gene imparts to the plant an increased resistance to, or tolerance of, pathogens, herbicides, insecticides and other biocides.
- 21. Process according to claim 19, wherein the said structural gene improves the formation and quality of reserve and stored substances in leaves, seeds, tubers, roots and stems.
- 22. Process according to claim 19, wherein the said structural gene codes for pharmaceutically acceptable active substances.
- 23. Process according to claim 17, wherein the said expression signals originate from genes of plants or plant viruses.
- 24. Process according to claim 17, wherein the transforming gene is composed of gene fragments from more than one strain, one variety or one species of the same organism.
- 25. Process according to claim 17, wherein the said transforming gene is composed both of genomic DNA, and of cDNA and/or synthetic DNA.
- 26. Process according to claim 17, wherein the transforming gene is composed of gene fragments from several organisms that belong to different genera.
- 27. Process according to claim 17, wherein the said transforming gene consists of genomic DNA, of a cDNA or of synthetic DNA.
- 28. Process according to claim 17, wherein the said expression signals originate from a plant gene that codes for the small subunit of ribulosebisphosphatecarboxylase or the chlorophyll a/b binding protein.
- 29. Process according to claim 17, wherein the said expression signals originate from genes of plant viruses.
- 30. Process according to claim 29, wherein the plant virus is Cauliflower Mosaic Virus (CaMV).
- 31. Process according to claim 29, wherein the expression signals are 35S expression signals of the CaMV genome.
- 32. Process according to claim 30, wherein the expression signals are the 19S expression signals of the CaMV genome.
- 33. A process for direct gene transfer into plant protoplasts that are amenable to transformation via direct gene transfer, which process comprising:
- isolating plant protoplasts from plant tissue and culturing said protoplasts in a nutrient media customarily used for culturing plant protoplasts;
- pre-incubating said protoplasts at a temperature of from 4.degree. to 10.degree. C. for from 20 minutes to 6 hours in a pre-incubation medium containing Ca.sup.2+, K.sup.+ and Na.sup.+ ions and a suitable carbon source before the actual transformation;
- separating said protoplasts from the pre-incubation medium and resuspending said protoplasts in a transformation medium which contains as an essential component from 10 to 30 mM Mg.sup.2+ ions in the presence or absence of Ca.sup.2+ ions;
- directly thereafter, adding a DNA sample to said transformation medium;
- from 0.1 to 10 minutes thereafter, adding polyethylene glycol (PEG) to said transformation medium; and
- incubating said protoplasts and DNA sample in said transformation medium for a period that is sufficient for the incorporation of said DNA into said protoplasts.
- 34. Process according to claim 33, wherein the final concentration of polyethylene glycol in the said incubation medium is from 10% to 30%.
Priority Claims (1)
Number |
Date |
Country |
Kind |
4866/86-8 4866/86 |
Dec 1986 |
CHX |
|
Parent Case Info
This application is a continuation of application Ser. No. 128,014, filed Dec. 2, 1987, now abandoned.
US Referenced Citations (1)
Number |
Name |
Date |
Kind |
4684611 |
Schilperoort et al. |
Aug 1987 |
|
Foreign Referenced Citations (1)
Number |
Date |
Country |
0193259 |
Sep 1986 |
EPX |
Continuations (1)
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Number |
Date |
Country |
Parent |
128014 |
Dec 1987 |
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