Abstract
A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l−1 kanamycin or 5 mg l−1 paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Brodelius, P. (1986) Immobilized plant cells and protoplasts. In: Evans, D. A., Sharp, W. R. and Ammirato, P. V. eds,Handbook of Plant Cell Culture. Vol 4 Techniques and Applications, pp. 287–315. New York: Macmillan
Caboche, M. (1980) Nutritional requirement of protoplast derived haploid tobacco cells grown at low densities in liquid medium.Planta 149, 7–18.
Capecchi, M.R. (1989) Altering the genome by homologous recombination.Science 244, 1288–92.
Crossway, A., Oakes, J.V., Irvine, J.M., Ward, B., Knauf, V. C. and Shewmaker, C.K. (1986) Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts.Mol. Gen. Genet. 202, 179–85.
Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem.,132, 6–13.
Gasser, C.S. and Fraley, R.T. (1989) Genetically engineering plants for crop improvement.Science 244, 1293–9.
Guerche, P., Bellini, P., Le Moullec, J.-M. and Caboche, M. (1987) Use of a transient expression assay for the optimisation of direct gene transfer into tobacco mesophyll protoplasts by electroporation.Biochim. 69, 621–8.
Hooykaas, P.J.J. (1989) Transformation of plant cells viaAgrobacterium.Plant Mol. Biol. 13, 327–36.
Jaenisch, R. (1988) Transgenic animalsScience 240, 1468–74.
Kao, K.N. and Michayluk, M. (1975) Nutritional requirement for growth ofVicia hajastana cells and protoplasts at very low population density in liquid media.Planta 126, 105–10.
Karesch, H., Bilang, R. and Potrykus, I. (1991)Arabidopsis thaliana: protocol for plant regeneration from protoplasts.Plant Cell Rep. 9, 575–8.
Lassner, M.W., Peterson, P., and Yoder, Y.I. (1989) Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny.Plant Mol. Biol. Rep. 7, 116–28.
Maliga, P., Breznovits, A. and Marton, L. (1973) Streptomycin-resistant plants from callus cultures of haploid tobacco.Nature 244, 29–30.
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982)Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
McDonnell, R.E., Clark, R.D., Smith, W.A. and Hinchee, M.A. (1987) A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues.Plant Mol. Biol. Rep. 5, 380–6.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant 15, 473–97.
Nagy, J.I. and Maliga, P. (1976) Callus induction and plant regeneration from mesophyll protoplasts ofN. sylvestris.Z. Pflanzenphysiol. 78, 453–5.
Neuhaus, G., Spangenberg, G., Mittelsten-Scheid, O. and Schweiger, H.G. (1987a) Transgenic rapeseed plants obtained by the microinjection of DNA into microsporederived embryoids.Theor. Appl. Genet. 75, 30–6.
Neuhaus, G., Kranz, E., Spangenberg, G. and Schweiger, H.G. (1987b) Transformation ofNicotiana tabacum andBrassica napus by microinjection into protoplasts.Eur. J. Cell. Biol. 43, 39.
Neuhaus, G. and Spangenberg, G. (1990) Plant transformation by microinjection techniques.Physiol. Plant 79, 213–7.
Paszkowski, J., Shillito, R.D., Saul, M., Mandak, V., Hohn, T., Hohn, B. and Potrykus, I. (1984) Direct gene transfer to plants.EMBO J. 3, 2717–22.
Paszkowski, J., Baur, M., Bogucki, A. and Potrykus, I. (1988) Gene targeting in plants.EMBO J. 7, 4021–6.
Paszkowski, J., Saul, M.W. and Potrykus, I. (1989) Plant gene vectors and genetic transformation: DNA-mediated direct gene transfer to plants. In: Schell, J. and Vasil, I.K. eds,Cell Culture and Somatic Cell Genetics of Plants. Vol. 6. Molecular Biology of Plant Nuclear Genes, pp. 52–68. San Diego: Academic Press.
Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986). Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with new plant expression vectors.Nucl. Acids Res. 14, 5857–68.
Portrykus, I. (1990) Gene transfer to cereals: an assessment.Bio/Technology 8, 535–42.
Potrykus, I. and Shillito, R.D. (1986) Protoplasts: isolation, culture, plant regeneration.Plant Mol. Biol. 118, 549–78.
Reich, T.J., Iyer, V.N. and Miki, B.L. (1986) Efficient transformation of alfalfa protoplasts by the intranuclear micro-injection of Ti plasmids.Bio/Technology 4, 1001–4.
Sanford, C.J. (1988) The biolistic process.TIBTECH 6, 299–302.
Schweiger, H.G., Dirk, J., Koop, H.U., Kranz, E., Neuhaus, G. and Spangenberg, G. (1987) Individual selection, culture and manipulation of higher plant cells.Theor. Appl. Genet. 73, 769–83.
Smidsrød, O. and Skjåk-Braek, G. (1990) Alginote as immobilization matrix for cells.TIBTECH 8, 71–8.
Spangenberg, G., Osusky, M., Oliveira, M.M., Freydl, E., Nagel, J., Pais, M.S. and Potrykus, I. (1990) Somatic hybridization by microfusion of defined protoplast pairs inNicotiana: morphological, genetic, and molecular characterization.Theor. Appl. Genet. 80, 577–87.
Steinbiss, H.H. and Stabel, P. (1983) Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow.Protoplasma 116, 223–7.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schnorf, M., Neuhaus-Url, G., Galli, A. et al. An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis. Transgenic Research 1, 23–30 (1991). https://doi.org/10.1007/BF02512993
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02512993