Stable co-transformation of maize protoplasts with gusA and neo genes

Abstract

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

“…Transformants that contained large numbers of barhybridizing fragments (R1, R5, and R21) also contained large numbers of fragments that hybridized to the gene encoding GUS ( Figures 5A and 5B). Similar observations using independent plasmids were reported using PEGmediated transformation of A188 x BMS protoplasts (Lyznik et al, 1989) and bombardment-mediated transformation of BMS suspension cells (Spencer et al, 1990).…”

“…Gordon-Kamm, unpublished results). The successful use of aminoglycosides, such as kanamycin, to identify transformants from cultured embryogenic cells may require optimization of several parameters, such as the stage of cell growth, concentration of antibiotic, and duration of exposure Zhang et al, 1988;Lyznik et al, 1989). The endogenous resistance to kanamycin and G418 observed in many embryogenic maize cultures may be a contributing factor in the frequent recovery of highly resistant callus lines that do not contain neo (R.J. Daines, unpublished results).…”

Histochemical analysis of CaMV 35S promoter-?-glucuronidase gene expression in transgenic rice plants

“…It has also been used in stably transformed cereal cells to drive the expression of genes providing resistance to antibiotics [6,18,22,23,24,19,12]. The activity demonstrated in cellular studies indicated its suitability for production of transgenic cereals and Rhodes et al [ 18] reported regeneration of maize plants from cells transformed with a chimeric neomycin phosphotransferase (npt-II) gene driven by the CaMV 35S promoter, transformed cells being selected from non-transformed cells by their resistance to the antibiotic kanamycin.…”

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker forAgrobacterium tumefaciens-Mediated Transformation of Maize

“…Concurrent with gene delivery methods, selectable marker development has been integral in developing efficient maize transformation. Kanamycin (Fromm et al, 1986;Rhodes et al, 1988;Lyznik et al, 1989) and hygromycin (Walters et al, 1992) were two of the earliest antibiotics used as selection agents in corn. The first herbicide used as a selection agent for maize transformation was the Glu analog phosphinothricin or, more commonly, the tripeptide bialaphos (lphosphinothricyl-l-alanyl-l-ananine), which contains phosphinothricin as the active ingredient (Fromm et al, 1990;Gordon-Kamm et al, 1990).…”