Summary
The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Abbreviations
- Aph:
-
aphidicolin
- ATF:
-
absolute transformation frequency
- BLM:
-
bleomycin
- CaMV:
-
cauliflower mosaic virus
- CAT:
-
chloramphenicol acetyl transferase
- CHO:
-
Chinese hamster ovary cells
- EF:
-
enhancement factor
- Nos:
-
nopaline synthase
- NPTII:
-
neomycin phosphotransferase II
- Ocs:
-
octopine synthase
- PEG:
-
polyethyleneglycol
- RTF:
-
relative transformation frequency
References
Ballas, N., N. Zakai, D. Friedberg & A. Loyter, 1988. Linear forms of plasmid are superior to supercoiled structures as active templates for gene expression in plant protoplasts. Plant Molec. Biol. 11: 517–527.
Bates, G.W., S.A. Carle & W.C. Piastuch, 1990. Linear DNA intro-duced into carrot protoplasts by electroporation undergoes liga-tion and recircularization. Plant Molec. Biol. 14: 899–908.
Benediktsson, I., F. Köhler & O. Schieder, 1991. Transient and stable expression of marker genes in cotransformedPetunia protoplasts in relation to X-ray and UV-irradiation. Transgenic Res. 1:38–44.
Benediktsson, I., C.P. Spampinato, C.S. Andreo & O. Schieder, 1994. Analysis of DNA polymerase activity inPetunia protoplasts treated with clastogenic agents. Physiol. Plant 90: 445–450.
Bianchi, N.O. & D.M. López-Larraza, 1991. DNA damage and repair induced by bleomycin in mammalian and insect cells. Envi-ronm. Molec. Mutagen. 17: 63–68.
Bohr, V.A., C.A. Smith, D.S. Okumoto & P.C. Hanawalt, 1985. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more different than in the genome overall. Cell 40: 359–369.
Burger, R.M., J. Peisach & S.B. Horwitz, 1981. Mechanism of bleomycin action:in vitro studies. Life Sci. 28: 715–727.
Chatterjee, A. & M.J. Raman, 1988. A comparison of abera-tion distribution and cell-cycle progression in cells treated with bleomycin with those exposed to X-rays. Mutat. Res. 202: 51–57.
Czernilowski, A.P., R. Hain, L. Herrera-Estrella, H. Lörz, E. Goy-vaerts, B.J. Baker & J. Schell, 1986. Fate of selectable marker DNA integrated into the genome ofNicotiana tabacum. DNA 5: 101–113.
Davey, M.R., E.C. Cocking, J. Freeman, N. Pearce & I. Tudor, 1980. Transformation ofPetunia protoplasts by isolatedAgrobacterium Plasmids. Plant Sci. Lett. 18: 307–313.
Dzelzkalns, V.A. & L. Bogorad, 1985. Stable transformation of the cyanobacteriumSynechocystis sp. PCC 6803 induced by irradia-tion. J. Bacteriol. 165 (3): 964–971.
Fraley, R.T., S.G. Rogers, R.B. Horsch, P.R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fry, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann & S.L. Woo, 1983. Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80: 4803–4807.
Gharti-Chhetri, G.B., W. Cherdshewasart, J. Dewulf, J. Paszkowski, M. Jacobs & I. Negrutiu, 1990. Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NTPII gene integration, its inheritance and factors affecting these processes inNicotiana plumbaginifolia. Plant Molec. Biol. 14: 687–696.
Gould, A.R. & R.J. Daines, 1985. Plant protoplasts and the cell cycle. In: L.C. Fowke & F. Constable (Eds). Plant Protoplasts, pp. 67–76. CRC-Press, Boca Raton, Florida.
Hain, R., P. Stabel, A.P. Czernilofsky, H.H. Steinbiß, L. Herrera-Estrella & J. Schell, 1985. Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant pro-toplasts. Mol. Gen. Genet. 199: 161–168.
Hall, R.D., F.A. Krens & J.A. Rouwendal, 1992. DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionizing radiation in fusion experi-ments? Physiol. Plant 85: 319–324.
Herrera-Estrella, L., M. De Block, E. Messens, J.-P. Hernalsteens, M. Van Montagu & J. Schell, 1983. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2; 6: 987–995.
Hobbs, S.L.A., T.D. Warkentin & C.M.O. DeLong, 1993. Trans-gene copy number can be positively or negatively associated with transgene expression. Plant Molec. Biol. 21: 17–26.
Hoffmann, F. & D. Hess, 1972. Die Aufnahme radioaktiv markierter DNS in isolierte Protoplasten vonPetunia hybrida. Z. Pflanzen-physiol. 69: 81–83.
Köhler, F., C. Golz, S. Eapen & O. Schieder, 1987. Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean. Plant Sci. 53: 87–91.
Köhler, F., G. Cardon, M. Pöhlmann, R. Gill & O. Schieder, 1989. Enhancement of transformation rates in higher plants by low-dose irradiation: are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Molec. Biol. 12: 189–199.
Köhler, F., I. Benediktsson, G. Cardon, C.S. Andreo & O. Schieder, 1990. Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer. Theor. Appl. Genet. 79: 679–685.
Krüger-Lebus, S. & I. Potrykus, 1987. A simple and efficient method for direct gene transfer toPetunia hybrida without electropora-tion. Plant Molec. Biol. Reporter 5 (2): 289–294.
Kuo, T.M., 1981. Preferential damage of active chromatin by bleomycin. Cancer Res. 41: 2439–2443.
Mellon, I., A.B. Vilhelm, C.A. Smith & P.C. Hanawalt, 1986. Pref-erential DNA repair of an active gene in human cells. Proc. Natl. Acad. Sci. USA 83: 8878–8882.
Meyer, P., E. Walgenbach, K, Bussmann, G. Hombrecher & H. Saedler, 1985. Synchronized tobacco protoplasts are efficiently transformed by DNA. Mol. Gen. Genet. 201 (3): 513–518.
Miller, C.K. & H.M. Temin, 1983. High efficiency ligation and recombination of DNA fragments by vertebrate cells. Science 220: 606–609.
Morgan, W.F. & J.E. Cleaver, 1983. Effect of 3-aminobenzamide on the rate of ligation during the repair of alkylated DNA in human fibroblasts. Cancer Res. 43: 3104–3107.
Nairn, R.S., G.M. Adair, C.B. Christmann & R.M. Humphrey, 1991. Ultraviolet stimulation of intermolecular homologous recombi-nation in Chinese hamster ovary cells. Mol. Carcinogen. 4: 519– 526.
Negrutiu, I., R. Shillito, I. Potrykus, G. Biasini & F. Sala, 1987. Hybrid genes in the analysis of transformation conditions. I. Set-ting up a simple method for direct gene transfer in plant proto-plasts. Plant Molec. Biol. 8: 363–373.
Paszkowski, J., R.D. Shillito, M. Saul, V. Mandák,T. Hohn, B. Hohn & I. Potrykus, 1984. Direct gene transfer to plants. EMBO J. 3: 2717–2722.
Paszkowski, J., M. Baur, A. Bogucki & I. Potrykus, 1988. Gene targeting in plants. EMBO J. 7: 4021–4026.
Paszkowski, J., A. Peterhans, H. Schlüpmann, C. Basse, E.G. Lebel & J. Masson, 1992. Protoplasts as tools for plant genome modi-fications. Physiol. Plant 85: 352–356.
Postel, E.H., 1985. Enhancement of genetic transformation frequen-cies of mammalian cell cultures by damage to the cell DNA. Mol. Gen. Genet. 201: 136–139.
Potrykus, I., 1991. Gene transfer to plants: Assessment of published approaches and results. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205–225.
Pröls, M., R. Töpfer, J. Schell & H.H. Steinbiß, 1988. Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance. Plant Cell Rep. 7: 221–224.
Puite, K.J., 1992. Progress in plant protoplast research. Physiol.Plant 85: 403–410.
Schocher, R.J., R.D. Shillito, M.W. Saul, J. Pazkowski & I. Potrykus, 1986. Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technology 4: 1093–1096.
Shillito, R.D., M.W. Saul, J. Paszkowski, M. Müller & I. Potrykus, 1985. High efficiency direct gene transfer to plants. Bio/Technology 3: 1099–1103.
Shimamoto, M., R. Terada, T. Izawa & H. Fujimoto, 1989. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274–276.
Spivak, G., A.K. Ganesan & P.C. Hanawalt, 1984. Enhanced trans-formation of human cells by UV irradiated pSV2 plasmids. Mol. Cell Biol. 4: 1169–1171.
Terleth, C., P. van de Putte & J. Brouver, 1991. New insights in DNA repair: preferential repair of transcriptionally active DNA. Mutagenesis 6: 103– 111.
Tyagi, S., B. Spörlein, A.K. Tyagi, R.G. Herrmann & H.U. Koop, 1989. PEG-and electroporation-induced transformation inNico-tiana tabacum: influence of genotype on transformation frequen-cy. Theor. Appl. Genet. 78: 287–292.
Umezawa, H., 1971. Natural and artificial bleomycins: chemistry and antitumor activities. Pure Appl. Chem. 28: 665–680.
Van Duin, M., A. Westerveld & J.H.J. Hoeijmakers, 1985. UV stim-ulation of DNA-mediated transformation of human cells. Mol. Cell Biol. 5 (4): 734–741.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Springer Science+Business Media New York
About this chapter
Cite this chapter
Benediktsson, I., Spampinato, C.P., Schieder, O. (1995). Studies of the mechanism of transgene integration into plant protoplasts: improvement of the transformation rate. In: Cassells, A.C., Jones, P.W. (eds) The Methodology of Plant Genetic Manipulation: Criteria for Decision Making. Developments in Plant Breeding, vol 3. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0357-2_6
Download citation
DOI: https://doi.org/10.1007/978-94-011-0357-2_6
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-4159-1
Online ISBN: 978-94-011-0357-2
eBook Packages: Springer Book Archive