Advertisement

PEG-Mediated Protoplast Transformation with Naked DNA

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 82)

Abstract

Direct introduction of DNA into plant protoplasts facilitates a rapid analysis of transient gene expression, as well as the generation of stably transformed transgenic plants. Transient gene expression assays performed after DNA transformation permit a comparative analysis of cis–actmg regulatory sequences and then function m transcriptional control of plant genes by signaling pathways mediating cellular responses to different environmental and hormonal stimuli (1). There are a number of methods for introducing DNA into plant protoplasts, but the most commonly used technique 1s the polyethylene glycol (PEG)–mediated DNA uptake. The PEG–mediated transformation is simple and efficient, allowing a simultaneous processing of many samples, and yields a transformed cell population with high survival and division rates (2). The method utihzes inexpensive supplies and equipments, and helps to overcome a hurdle of host range limitations of Agrobactenum–mediated transformation The PEG–mediated DNA transfer can be readily adapted to a wide range of plant species and tissue sources.

Keywords

Green Fluorescence Protein Green Fluorescence Protein Fluorescence Transient Gene Expression Plant Protoplast Protoplast Suspension 
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.
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Morgan, M. K. and Ow, D W (1995) Polyethylene glycol–mediated transformation of tobacco leaf mesophyl protoplasts. an experiment in the study of Cre–Lox recombmation, in Methods zn Plant Molecular Biology A Laboratory Course Manual (Mahga, P, Klessig, D F., Cashmore, A R, Gnussem, W, and Varner, J. E eds,), Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1–17.Google Scholar
  2. 2.
    Potrykus, I (1991) Gene transfer to plants assessment of pubhshed approaches and results Ann Rev Plant Physlol Plant Mol Blol 42,205–255CrossRefGoogle Scholar
  3. 3.
    Morris, P. C and Altmann, T (1994) Tissue culture and transformatton, in Arabidopszs (Meyerowttz, E. M and Somerville, C. R., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 173–222Google Scholar
  4. 4.
    Damm, B, Schmidt, R, and Willmitzer, L. (1989) Effictent transformation of Arabidopszs thaltana using direct gene transfer to protoplasts Mol Gen Genet 217,6–12.PubMedCrossRefGoogle Scholar
  5. 5.
    Karesch, H., Btlang, R., Mtttelsten-Schetd, O., and Potrykus, I. (1991) Direct gene transfer to protoplasts of Arabldopsls thaliana Plant Cell Rep 9, 575–578Google Scholar
  6. 6.
    Mathur, J., Szabados, L., and Koncz, C. (1995) A simple method for isolation, liquid culture, transformation and regenerationof Arabldopszs thalzana protoplasts. Plant Cell Rep 14, 221–226.Google Scholar
  7. 7.
    Negrutm, I., Shillito, R., Potrykus, I., Biasini, G., and Sala, F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer m plant protoplasts. Plant Mol. Blol 8, 363–373CrossRefGoogle Scholar
  8. 8.
    Chalfie M Tu, Y. Euskirchen, G., Ward, W. W., and Prasher, D C. (1994) Green fluoresence protein as a marker for gene expression Science 263, 802–805.PubMedCrossRefGoogle Scholar
  9. 9.
    Helm, R., Prasher, D C, and Tsien, R. Y. (1994) Wavelength mutatrons and posttranslattonal autoxrdatron of green fluorescent protein. Proc Nat 1 Acad Scz USA 91, 12501–12504.CrossRefGoogle Scholar
  10. 10.
    Murashige, T. and Skoog, F. (1962) A revised medium for raped growth and bioassay with tobacco tissue cultures. Physzol Plant 15,473–497.CrossRefGoogle Scholar
  11. 11.
    Gamborg, 0. L., Miller, R A, and OJtma, K (1968) Nutrrent requirements of suspensions cultures of soybean root cells. Exp Cell Rex 50, 151–158.CrossRefGoogle Scholar
  12. 12.
    Koncz, C., Schell, J, and Rtdet, G. P (1992) T-DNA transformatton and insertronal mutagenesis, in Methods in Arabidopszs Research (Koncz, C., Chua, N.-H., and Schell, J., eds.), World Scientific, Singapore, pp 224–273Google Scholar
  13. 13.
    Jefferson R A (1987) Assaymg chrmerrc genes m plants: the GUS gene fusion system Plant Mel BIOI Rep 5,387–405CrossRefGoogle Scholar
  14. 14.
    Haseloff, J. and Amos, B (1995) GFP in plants. Trends Genet 11, 328–329PubMedCrossRefGoogle Scholar
  15. 15.
    Mason, J and Paszkowski, J. (1992) The culture response of Arab1dop.m thalzana protoplasts 1s determined by the growth conditrons of donor plants. Plant J 2, 829–833.CrossRefGoogle Scholar
  16. 16.
    Rasmussen, J. O. and Rasmussen, 0.S. (1993) PEG-medrated uptake and transient GUS expressron in carrot, rapeseed and soybean protoplasts. Plant Scz 89,199–2CrossRefGoogle Scholar
  17. 17.
    Rerchel, C., Mathur, J, Langenkemper, C., Eckes, P, Koncz, C., Rers. B, Schell, J., and Maas, C (1996) Enhanced green fluorescence by the expression of an Aequorea vzctorza GFP mutant m mono-and drcotyledonousplant cells. Proc Nat Acad Scl USA 93, 5888–5893.CrossRefGoogle Scholar

Copyright information

© Human press Inc, Totowa, NJ 1998

Authors and Affiliations

  1. 1.Max, Planck-Institut fur ZuchtungsforschungCologneGermany

Personalised recommendations

Cite protocol

Buy options