Transient and stable electrotransformations of intact black Mexican sweet maize cells are obtained after preplasmolysis

Abstract

When interested in plant cell transformation, the cell wall is often considered as a barrier to DNA transfer, which is only overcome by wounding or wall degrading enzymes. In this work, we demonstrate that cell plasmolysis before electropulsation is an efficient approach to DNA delivery into intact plant cells. Using such a method, transient expression (β-glucuronidase and chloramphenicol acetyltransferase) and stable expression (phosphinotricin acetyltransferase) of exogenous genes are obtained in intact black Mexican sweet maize cells.

Electroporation in Biological Cell and Tissue: An Overview

“…These partial detachments of the cell wall from the membrane cause a void space between the rigid cell wall and the cell membrane and enables the required contact between the cell membrane and the macromolecule that is being introduced into the cytoplasm (Fig. 5) (D'Halluin et al 1992;Ganeva et al 1995;Sabri et al 1996a;Eynard et al 1997;Wu and Feng 1999).…”

“…Even though the cell wall represents a barrier, osmotic shock pretreatment that provokes plasmolysis can be used to create a passage of molecules through the cell wall (D'Halluin et al 1992;Ganeva et al 1995;Sabri et al 1996a;Eynard et al 1997;Wu and Feng 1999).…”

“…To improve the transport of macromolecules through the cell wall of bacteria, yeast, and plant cells, different pretreatments were suggested. Before electroporation, partial disruption of the cell wall was effective for bacteria and yeast, while for plant cells, pre-pulse plasmolysis was successfully applied (D'Halluin et al 1992;Ganeva et al 1995;Sabri et al 1996a;Eynard et al 1997;Wu and Feng 1999).…”

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

“…Electroporation conditions were: 500 µF, 100 V.cm -1 , 20 Ω for buffer I and III, 50 Ω for buffer II, 10 µg of supercoiled DNA. Bars indicate standard errors and small letters indicate significant differences at p < 0.05. transformation efficiency using linear plasmid DNA has been reported for bacteria (Rittich and Spanova, 1996), yeasts and filamentous fungi (Kwon-Chung et al, 1998), plant protoplasts (Lin et al, 1997) and intact tissue (Akella and Lurquin, 1993;Dillen et al, 1995;Lin et al, 1997;Sabri et al, 1996;Saunders et al, 1995). Due to the inner twist of the molecule and to the absence of distortions imposed on the double helix structure (Drew et al, 1988), linear plasmid DNA is highly mobile under electric fields (Courey and Wang, 1983).…”

Transient gene expression in electroporated intact tissues of Stylosanthes guianensis (Aubl.) Sw.

“…Electric pulses elevate the transmembrane potential promoting pore formation due to increased dipole moment of the hydrophilic lipid heads (Neumann et al, 1982;Kinosita & Tsong, 1977). The effects of an electric field on cell walls are still unknown although transgene delivery and expression have been described for goat's rue embryos (Collen & Jarl, 1999), intact cells of maize (Songstad et al, 1993;Sabri et al, 1996), common bean, cowpea, and other grain legumes (Akella & Lurquin, 1993;Dillen et al, 1995), rice and wheat (Dekeyser et al, 1990;Xu & Li, 1994), alfalfa (Senaratna et al, 1991) and sugar beet (Lindsey & Jones, 1987).…”