Transient Gene Expression in Intact and Organized Rice Tissues

Abstract

Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a [beta]-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light-harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.

Ascorbate Increases Electrotransformation Efficiency of Intact Maize Cells

“…The source of the enzymes are microorganisms and in some instances protein engineering to improve stability at elevated temperatures has been reported (1). Be it the screening procedure to obtain potential organism or mutational analysis of cloned gene (2), assays at elevated temperatures for the enzyme in crude culture broth are affected by interfering enzymes and proteases. Gene expression studies using heterologous hosts defining mutations must also take into account the above-stated interferences.…”

“…A difficulty is nevertheless present concerning the transfer of plasmids across the cell wall. It has been proposed to facilitate the transfer by a damage of the wall either by mechanical (1,2) or by proteolytic means (3). Such stresses may generate a defense mechanism of the cell, which was not evaluated.…”

Asiatic Beans

“…Owing to the natural resistance of many plants including legumes to antibiotics (Dekeyser et al, 1990), appropriate antibiotic and its effective concentration for selection of transformants for particular Vigna species can only be determined empirically (Babaoglu et al, 2000). The dose of selective agent (lethal or sublethal), time of its application (i.e., more precisely immediately after co-culture or at a much delayed stage), and increments (gradual increasing or decreasing increments) need to be carefully assessed for a particular species and a variety of explants.…”

Molecular improvement of cereals

“…Transient expression as well as stable integration of the introduced genes has been demonstrated [19,52,93], and the procedure has also been used successfully to obtain transgenic plants by delivering D N A into enzymatically or mechanically wounded immature embryos as well as the more common type I callus tissues of maize [23] or into bisected mature embryos of rice [114, Table 1]. Electroporation is a simple, rapid and inexpensive procedure that does not require elaborate equipment.…”