Skip to main content
SpringerLink
Log in

Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook

  • Published:
Plant Cell, Tissue and Organ Culture Aims and scope Submit manuscript

Abstract

Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm-1 (1000 μs), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 μg ml-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BAP:

6-benzylaminopurine

CAT:

chloramphenicol acetyltransferase

CPW 13M:

CPW salts medium with 13% (w/v) mannitol

DC:

direct current

FDA:

fluorescein diacetate

f. wt:

fresh weight

GUS:

β-glucuronidase

K:

Kao (1977)

MES:

2-N-morpholinoethane sulfonic acid

MS:

Murashige & Skoog (1962)

NAA:

α-naphthaleneacetic acid

PVP-10:

polyvinylpyrrolidone (Av MW 10,000)

TLC:

thin layer chromatography

References

  • Bekkaoui F, Pilon M, Laine E, Raju DDS, Crosby WL & Dunstan DI (1988) Transient gene expression in electroporated Picea glauca protoplasts. Plant Cell. Rep. 7: 481–484

    Article  Google Scholar 

  • Charest PJ & Klimaszewska K (1990) Transient expression of foreign chimeric genes in hybrid larch (Larix eurolepis) protoplasts. J. Cell Biochem. Sup. 14E: 277

    Google Scholar 

  • Gilmour DM, Davey MR & Cocking EC (1989) Production of somatic hybrid tissues following chemical and electrical fusion of protoplasts from albino cell suspensions of Medicago sativa and M. borealis. Plant Cell Rep. 8: 29–32

    Google Scholar 

  • Golds TJ, Davey MR, Rech EL & Power JB (1990) Methods of gene transfer and analysis in higher plants. In: Pollard JW & Walker JM (Eds) Methods in Molecular Biology, Vol 6 (pp 341–371). Ch 33 Plant Cell and Tissue Culture, Humana Press

  • Gorman CM, Moffat LF & Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2: 1044–1051

    PubMed  Google Scholar 

  • Gupta PK, Dandekar AM & Durzan DJ (1988) Somatic pro-embryo formation and transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts. Plant Sci. 58: 85–92

    Article  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405

    Google Scholar 

  • Joersbo M & Brunstedt J (1990) Direct gene transfer to plant protoplasts by electroporation by alternating, rectangular and exponentially decaying pulses. Plant Cell. Rep. 8: 701–705

    Article  Google Scholar 

  • Kao KN (1977) Chromosomal behaviour in somatic hybrids of soybean-Nicotiana glauca L. Mol. Gen. Genet. 150: 225–230

    Article  Google Scholar 

  • Krens FA, Molendijk L, Wullems GJ & Schlperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72–74

    Google Scholar 

  • Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497

    Google Scholar 

  • Pietrzak M, Shillito RD, Hohn T & Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Res. 14: 5857–5868

    PubMed  Google Scholar 

  • Power JB, Davey MR, McLellan & Wilson D (1989) Laboratory Manual: Plant Tissue Culture. Plant Genetic Manipulation Group, Dept. of Botany, University of Nottingham, UK

    Google Scholar 

  • Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning. A Laboratory Manual. Second Edition, Vol 1: 33–41 Cold Spring Harbour Laboratory Press. USA

    Google Scholar 

  • Santos AVP, d'Utra Vaz FB & Power JB (1990) Towards somatic hybridization in the Genus Eucalyptus. Abstracts VIIth International Congress on Plant Tissue and Cell Culture, Amsterdam: 208

  • Séguin A & Lalonde M (1988) Gene transfer by electroporation in Betulaceae protoplasts (Alnus incana). Plant Cell Rep. 7: 367–370

    Google Scholar 

  • Shillito RD, Saul MW, Paszkowski J, Müller M & Potrykus I (1985) High efficiency direct gene transfer to plants. Bio/Technol. 3: 1099–1103

    Article  Google Scholar 

  • Tautorus TE, Bekkaoui F, Pilon M, Datla RSS, Crosby WL, Fowke LC & Dunstan DI (1989) Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts. Theor. Appl. Genet. 78: 531–536

    Article  Google Scholar 

  • Teullères C, Grima-Pettenati J, Curie C, Teissic J & Boudet AM (1991) Transient gene expression in protoplasts and intact cells of Eucalyptus gunnii after electrical or chemical treatment. Plant Cell Tiss. Org. Cult. 25: 125–132

    Google Scholar 

  • Watts JW & King JM (1984) A simple method for large-scale electrofusion and culture of plant protoplasts. BioSci Rep. 4: 335–342

    PubMed  Google Scholar 

  • Wilson SM, Thorpe TA & Moloney MM (1989) PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca. Plant Cell Rep. 7: 704–707

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, NG7 2RD, Nottingham, UK

    G. Manders, F. B. d'Utra Vaz, M. R. Davey & J. B. Power

  2. Universidade Federal de Alagoas, Rio Largo, 57100, Alagoas, Brazil

    A. V. P. dos Santos

Authors
  1. G. Manders
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. V. P. dos Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. B. d'Utra Vaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. R. Davey
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. B. Power
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Manders, G., dos Santos, A.V.P., d'Utra Vaz, F.B. et al. Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook. Plant Cell Tiss Organ Cult 30, 69–75 (1992). https://doi.org/10.1007/BF00040003

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00040003

Key words

Search

Navigation