We have investigated the basis for increased transient reporter gene expression following electroporation of protoplasts from uniform carrot cell suspension cultures at increasing DNA concentrations. Use of a combination of histochemical and fluorometric GUS gene assays allowed differentiation between increases due to a higher proportion of expressing protoplasts and increases due to higher expression by each expressing protoplast. A plateau of 20–25% expressing protoplasts was reached by 50 μg ml−1 DNA but total expression continued to increase in direct proportion to applied DNA concentration up to at least 100 μg ml−1. This indicates the existence of a subpopulation of protoplasts competent for the uptake and expression of genes by electroporation.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Fromm M, Loverine P, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci USA 82:5824–5828.
Fromm M, Walbot V (1987) Transient expression of DNA in plant cells. In: Hohn TL, Schell J (Eds) Plant DNA Infectious Agents, Springer Verlag Wien NY. pp 303–309.
Gallie DR, Lucas WJ, Walbot V (1989) Visualizing mRNA expression in plant protoplasts: Factors influencing efficient mRNA uptake and translation. The Plant Cell 1:301–311.
Huang YW, Dennis ES (1989) Factors affecting stable transformation of maize protoplasts by electroporation. Plant Cell Tiss. and Org. Cult. 18:281–298.
Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5:387–405.
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907.
Langdridge WHR, Li BJ, Szalay AA (1985) Electric field mediated stable transformation of carrot protoplasts with naked DNA. Plant Cell Rep. 4:355–359.
Langdridge WHR, Li BJ, Szalay AA (1987) Uptake of DNA and RNA into cells mediated by electroporation. Meth. Enz. 153:336–350.
Larkin PJ, Taylor BH, Gersmann M, Brettell RIS (1990) Direct gene transfer to protoplasts. Aust. J. Plant Physiol. In press.
Lindsay K, Jones MGK (1987) Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol. Biol. 10:43–53.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473–479.
Negrutiu I, Mouras A, Horth M, Jacobs M (1987) Direct gene transfer to plants: Present developments and some future prospectives. Plant Physiol. Biochem. 25:493–503.
Nishiguchi M, Sato T, Motoyushi F (1987) An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles. Plant Cell Rep. 6:90–93.
Okada K, Takebe I, Nagata T (1986) Expression and integration of genes introduced into highly synchronised plant protoplasts. Mol. Gen. Genet. 205:398–403.
Ou-Lee T, Turgeon R, Wu R (1986) Expression of a foreign gene linked to either a plant-virus as a Drosophila promoter, after electroporation of protoplasts of rice, wheat and sorghum. Proc. Natl. Acad. Sci USA 83:6815–6819.
Potrykus I, Paszkowski J, Saul WM, Negrutiu I, Shillito R (1987a) Direct gene transfer to plants: Facts and future. In: Green CE et al. (eds) Plant Tissue and Cell Culture, Allen R. Liss, Inc. NY. pp 289–302.
Potrykus I, Paszkowski J, Shillito RD, Saul MW (1987b) Direct gene transfer to plants. In: Hohn TL, Schell J (eds) Plant DNA Infectious Agents, Springer Verlag Wien NY. pp 229–247.
Pröls M, Töpfer R, Schell J, Steinbill H (1988) Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance. Plant Cell Rep. 7:221–224.
Schocher RJ, Shillito RD, Saul MW, Paszkowski J, Potrykus I (1986) Co-transformation of foreign genes into plants. Bio/Tech. 4:1093–1096.
Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Bio/Tech. 3:1099–1103.
Taylor BH, Larkin PJ (1988) Analysis of electroporation efficiency in plant protoplasts. Aust. J. of Biotech. 1:52–57.
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Tech. 47:189–194.
Zhang HM, Yang H, Rech EL, Golds TJ, Davis AS, Mulligan BJ, Cocking EC, Davey MR (1988) Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep. 7:379–384.
Communicated by C. Quiros
About this article
Cite this article
Bower, R., Birch, R.G. Competence for gene transfer by electroporation in a sub-population of protoplasts from uniform carrot cell suspension cultures. Plant Cell Reports 9, 386–389 (1990). https://doi.org/10.1007/BF00232405
- Gene Expression
- High Expression
- Gene Transfer
- Reporter Gene
- Suspension Culture