Microinjection: An Experimental Tool for Studying and Modifying Plant Cells

  • Brian L. A. Miki
  • Terry J. Reich
  • V. N. Iyer
Part of the Plant Gene Research book series (GENE)


The conviction that DNA is the genetic material of cells has its origins in a set of experiments in which purified DNA was transferred and expressed in bacterial cells. It is therefore no surprise that since these initial experiments with bacteria, methods of transferring DNA and other molecules into eukaryotic cells have been explored with varying degrees of success and usefulness. The most direct way of delivering a population of molecules into a cell or cell compartment is to microinject it mechanically (Celis, 1984; Celis et al., 1986). Techniques to accomplish this have been developed for frog oocytes (Gurdon et al., 1971; Kressman et al., 1978; Rusconi and Schaffner, 1981), insect embryos (Rubin and Spradling, 1982), animal egg cells (Brinster et al., 1985; Gordon et al., 1980; Hammer et al., 1985; Wagner et al., 1981) and mammalian somatic cells (Capecchi, 1980; Diacumakos, 1973; Graessman et al., 1980a, b). The anatomical organization of plant cells and the experimental difficulties associated with their manipulation in culture prevented a facile transposition of such techniques from animal to plant cells (Steinbiss et al., 1984). Fortunately, this situation is changing rapidly. It is now possible to microinject some plant cells and various protoplast types successfully (Crossway et al., 1986; Reich et al., 1986 a, b, c). It is the intent of this contribution to review these methodologies so that they can be extended, improved upon and become easily accessible to the community of plant cell and molecular biologists. Through this discussion we hope that the experimental advantages offered by microinjection become apparent.


Plant Cell Mesophyll Protoplast Lucifer Yellow Plant Protoplast Tobacco Protoplast 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ammirato, P. V., 1983: Embryogenesis. In: Evans, D. A., Sharp, W. R., Ammirato, P. V., Yamada, Y. (eds.), Handbook of plant cell culture (Vol. 1 ), pp. 82–123. New York: Macmillan Publishing Co.Google Scholar
  2. Bar-Sagi, D., Feramisco, J. R., 1985: Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell 42, 841–848.PubMedCrossRefGoogle Scholar
  3. Berkenstam, A., Ahlberg, J., Glaumann, H., 1986: Lysosomal uptake of isolated cell organelles microinjected into Hela cells. Exp. Cell Res. 163, 301–308.PubMedCrossRefGoogle Scholar
  4. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D., 1985: Factors affecting the efficiency of introducing foreign DNA into mice by mi- croinjecting eggs. Proc. Natl. Acad. Sci., U.S.A. 82, 4438–4442.PubMedCrossRefGoogle Scholar
  5. Capecchi, M. R., 1980: High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488.PubMedCrossRefGoogle Scholar
  6. Celis, J. E., 1984: Microinjection of somatic cells with micropipettes: comparison with other transfer techniques. Biochem. J. 223, 281–291.PubMedGoogle Scholar
  7. Celis, J. E., Graessman, A., Loyter, A., 1986: Microinjection and organelle trans-plantation techniques. London - Orlando: Academic Press.Google Scholar
  8. Crossway, A., Oakes, J. V., Irvine, J. M., Ward, B., Knauf, V. C., Shewmaker, C. K, 1986: Integration of foreign DNA following microinjection of tobacco meso- phyll protoplasts. Mol. Gen. Genet. 202, 179–185.CrossRefGoogle Scholar
  9. Deshayes, A., Herrera-Estrella, L., Caboche, M., 1985: Liposome-mediated trans-formation of tobacco mesophyll protoplasts by an Escherichia coli plasmid. EMBO J. 4, 2731–2737.PubMedGoogle Scholar
  10. Diacumakos, E. G., 1973: Methods for micromanipulation of human somatic cells in culture. Methods Cell Biol. 7, 287–311.PubMedCrossRefGoogle Scholar
  11. Dunwell, J. M., 1985: Anther and ovary culture. In: Bright, S. W. J., Jones, M. G. K. (eds.), Cereal tissue and cell culture (Advances in Agricultural Biotechnology), pp. 1–44. Dordrecht - Boston - Lancaster: Martinus Nijhoff - Dr. W. Junk Publishers.Google Scholar
  12. Evans, D. A., Bravo, J. E., 1983: Protoplast isolation and culture. In: Evans, D. A., Sharp, W. R., Ammirato, P. V., Yamada, Y. (eds.), Handbook of plant cell culture, pp. 124–176. New York: Macmillan Publishing Co.Google Scholar
  13. Firoozabady, E., Galbraith, D. W., 1984: Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens. Plant Cell Tissue Organ Culture 3, 175–188.CrossRefGoogle Scholar
  14. Flavell, R., Mathias, R., 1984: Prospects for transforming monocot crop plants. Nature (London) 307, 108–109.CrossRefGoogle Scholar
  15. Fraley, R. T., Rogers, S. G., Horsch, R. B., Eichholtz, D. A., Flick, J. S., Fink, C. L., Hoffman, N. L., Sanders, P. R., 1985: The SEV system: A new disarmed Ti plasmid vector system for plant transformation. Bio/Technology 3, 629–635.CrossRefGoogle Scholar
  16. Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Gallupi, G. R., Goldberg, S. B., Hoffmann, N. L., Woo, S. C., 1983: Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci., U.S.A. 80, 4803–4807.PubMedCrossRefGoogle Scholar
  17. Goodwin, P. B., Erwee, 1984: Intercellular transport studied by microinjection methods. In: Robards, A. W. (ed.), Botanical Microscopy. 1985, pp. 335–358.,Oxford - New York - Tokyo: Oxford University Press.Google Scholar
  18. Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H., 1980: Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77, 7380–7384.PubMedCrossRefGoogle Scholar
  19. Gordon-Kamm, W. J., Bushnell, W. R., 1985: Intranuclear microinjection of isolated protoplasts using differential interference contrast microscopy. (Abstracts of the first international congress of plant molecular biology, Savannah 1985), pp. 109. Athens: The University of Georgia Center for Continuing Education.Google Scholar
  20. Gould, A. R., Ashmore, S. E., 1982: Interaction of purified DNA with plant proto-plasts of different cell cycle stage: The concept of a competent phase for plant cell transformation. Theor. Appl. Genet. 64, 7–12.CrossRefGoogle Scholar
  21. Graessman, A., Graessman, M., Mueller, C., 1980a: Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 65, 816–825.CrossRefGoogle Scholar
  22. Graessmann, A., Wolf, H., Bornkamm, G. W., 1980b: Expression of Epstein-Barr viral genes in different cell types after microinjection of viral DNA. Proc. Natl. Acad. Sci., U.S.A. 77, 433–436.CrossRefGoogle Scholar
  23. Gurdon, J. B., Lane, C. D., Woodland, H. R., Marbaix, G., 1971: Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature (London) 233, 177–182.CrossRefGoogle Scholar
  24. Halliwell, R. S., Gazaway, W. S., 1975: Quantity of microinjected tobacco mosaic virus required for infection of single cultured tobacco cells. Virology 65, 583–587.PubMedCrossRefGoogle Scholar
  25. Hammer, R. E., Pursel, V. G., Rexroad, C. E., Wall, R. J., Bolt, D. J., Ebert, K. M., Palmiter, R. D., Brinster, R. L., 1985: Production of transgenic rabbits, sheep and pigs by microinjection. Nature (London) 315, 680–683.CrossRefGoogle Scholar
  26. Heberle-Bors, E., 1985: In vitro haploid formation from pollen: a critical review. Theor. Appl. Genet. 71, 361–374.CrossRefGoogle Scholar
  27. Herrera-Estrella, L., DeBlock, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M., Schell, J., 1983: Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2, 987–995.PubMedGoogle Scholar
  28. Holbrook, L. A., Reich, T. J., Iyer, V. N., Haffner, M., Miki, B. L., 1985: Induction of efficient cell division in alfalfa protoplasts. Plant Cell Rep. 4, 229–232.CrossRefGoogle Scholar
  29. Jones, M. G. K., 1985: Cereal protoplasts. In: Bright, S. W. J., Jones, M. G. K. (eds.), Cereal tissue and cell culture (Advances in Agricultural biotechnology), pp. 204–230. Dordrecht - Boston - Lancaster: Martinus Nijhoff - Dr. W. Junk Publishers.Google Scholar
  30. Kreis, T. E., Birchmeier, W., 1982: Microinjection of fluorescently labelled proteins into living cells with emphasis on cytoskeletal proteins. Int. Rev. Cytol. 75, 209–227.PubMedCrossRefGoogle Scholar
  31. Krens, F. A., Mans, R. M. W., van Slogteren, T. M. S., Hoge, J. H. C., Wullems, G. J., Schilperoort, R. A., 1985: Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti plasmids DNA: co-transfer of T-DNA and non T-DNA sequences. Plant Mol. Biol. 5, 223–234.Google Scholar
  32. Krens, F. A., Molendijk, L., Wullems, G. J., Schilperoort, R. A., 1982: In vitro trans-formation of plant protoplasts with Ti-plasmid DNA. Nature (London) 296, 72–74.CrossRefGoogle Scholar
  33. Kressmann, A. Clarkson, S. G., Telford, J. L., Birnstiel, M. L., 1978: Transcription of Xenopus tDNA1 met and sea urchin histone DNA injected into the Xenopus oocyte nucleus. Cold Spring Harbor Symp. Quant. Biol. 42, 1077–1082.PubMedGoogle Scholar
  34. Lawrence, W. A., Davies, D. R., 1985: A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep. 4, 33–35.CrossRefGoogle Scholar
  35. Lin, P.-F., Ruddle, F. H., 1981: Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads. Exp. Cell Res. 134, 485–488.PubMedCrossRefGoogle Scholar
  36. Maddock, S. E., 1985: Cell culture, somatic embryogenesis and plant regeneration in wheat, barley, oats, rye and triticale. In: Bright, S. W. J., Jones, M. G. K. (eds.), Cereal tissue and cell culture (Advances in agricultural biotechnology), pp. 131–174. Dordrecht - Boston - Lancaster: Martinus Nijhoff - Dr. W. Junk Publishers.Google Scholar
  37. Mangeat, P. H., Burridge, K., 1985: Microinjection and immunofluorescence microscopy as tools to study cytoskeletal organization in tissue culture cells. Techn. Immunocytochem. 3, 79–96.Google Scholar
  38. Mariotti, D., Davey, M. R., Draper, J., Freeman, J. P., Cocking, E. C., 1984: Crown gall tumorigenesis in the forage legume Medicago sativa L. Plant Cell Physiol. 25, 473–482.Google Scholar
  39. Matthews, J. A., Brown, J. W. S., Hall, T. C., 1981: Phaseolin mRNA is translated to yield glycosylated polypeptides in Xenopus oocytes. Nature (London) 294, 175–176.CrossRefGoogle Scholar
  40. Meyer, P., Walgenbach, E., Bussmann, K., Hombrecher, G., Saedler, H., 1985: Syn-chronized tobacco protoplasts are efficiently transformed by DNA. Mol. Gen. Genet. 201, 513–518.CrossRefGoogle Scholar
  41. Morikawa, H., Yamada, Y., 1985: Capillary microinjection into protoplasts and intranuclear localization of injected materials. Plant Cell Physiol. 26, 229–236.Google Scholar
  42. Nims, R. C., Halliwell, R. S., Rosberg, D. W., 1967a: Disease development in cultured cells of Nicotiana tabacum L. var. Samsun NN injected with tobacco mosaic virus. Cytologia 32, 224–235.Google Scholar
  43. Nims, R. C., Halliwell, R. S., Rosberg, D. W., 1967b: Wound healing in cultured tobacco cells following microinjection. Protoplasma 64, 305–314.CrossRefGoogle Scholar
  44. Palevitz, B. A., Hepler, P. K., 1985: Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow. Planta 164, 473–479.CrossRefGoogle Scholar
  45. Paszkowski, J., Shillito, R. D., Saul, M., Mandäk, V., Hohn, T., Hohn, B., Potrykus, I., 1984: Direct gene transfer to plants. EMBO J. 3, 2717–2722.PubMedGoogle Scholar
  46. Reich, T. J., Iyer, V. N., Haffner, M., Holbrook, L. A., Miki, B. L., 1986a: The use of fluorescent dyes in the microinjection of alfalfa protoplasts. Can. J. Bot. 64, 1259–1267.CrossRefGoogle Scholar
  47. Reich, T. J., Iyer, V. N., Miki, B. L., 1986b: Efficient transformation of alfalfa protoplasts by the intranuclear microinjection of Ti plasmids. Bio/Technology 4, 1001–1004.CrossRefGoogle Scholar
  48. Reich, T. J., Iyer, V. N., Scobie, B., Miki, B. L., 1986c: A detailed procedure for the intranuclear microinjection of plant protoplasts. Can. J. Bot. 64, 1255–1258.CrossRefGoogle Scholar
  49. Rubin, G. M., Spradling, A. C., 1982: Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353.PubMedCrossRefGoogle Scholar
  50. Rusconi, A., Schaffner, W., 1981: Transformation of frog embryos with a rabbit ß- globin gene. Proc. Natl. Acad. Sei., U.S.A. 78, 5051–5055.CrossRefGoogle Scholar
  51. Shillito, R. D., Paszkowski, J., Potrykus, I., 1983: Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep. 2, 244–247.CrossRefGoogle Scholar
  52. Shillito, R. D., Saul, M. W., Paszkowski, J., Müller, M., Potrykus, I., 1986: High efficiency direct gene transfer to plants. Bio/Technology 3, 1099–1103.CrossRefGoogle Scholar
  53. Steinbiss, H.-H., Stabel, P., 1983: Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer yellow. Protoplasma 116, 223–227.CrossRefGoogle Scholar
  54. Steinbiss, H.-H., Stabel, P., Töpfer, R., Hirtz, R. D., Schell, J., 1984: Transformation of plant cells by microinjection of DNA. In: Experimental manipulation of ovule tissue: their manipulation, tissue culture and physiology (Proceedings of the 1984 Wye International Symposium, Wye College, University of London, 1984), pp. 64–75. London: England.Google Scholar
  55. Van Lijsebettens, M., Inze, D., Schell, J., Van Montagu, M., 1986: Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefadens. J. Mol. Biol. 188, 129–145.PubMedCrossRefGoogle Scholar
  56. Wagner, T. E., Hoppe, P. C., Jollick, J. D., Scholl, D. R., Hodinka, R. L., Gault, J. B., 1981: Microinjection of a rabbit ß-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sei., U.S.A. 78, 6376–6380.CrossRefGoogle Scholar
  57. Wehland, J., Osborn, M., Weber, K., 1980: Phalloidin associates with microfilaments after microinjection into tissue culture cells. Eur. J. Cell Biol. 21, 188–194.PubMedGoogle Scholar
  58. Wehland, J., Weber, K., 1981: Actin rearrangement in living cells revealed by mi-croinjection of a fluorescent phalloidin derivative. Eur. J. Cell Biol. 24, 176–183.PubMedGoogle Scholar
  59. Zhou, G.-Y., Weng, J., Zeng, Y., Huang, J., Qian, S., Liu, G., 1983: Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 101, 433–481.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag/Wien 1987

Authors and Affiliations

  • Brian L. A. Miki
    • 1
  • Terry J. Reich
    • 2
  • V. N. Iyer
    • 2
  1. 1.Genetic Engineering SectionPlant Research Centre, Agriculture CanadaOttawaCanada
  2. 2.Department of BiologyCarleton UniversityOttawaCanada

Personalised recommendations