Stable transformation of tobacco by electroporation: evidence for plasmid concatenation.

Abstract

Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence ofthe linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 x 10-(based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-pis-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots ofDNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head-to-head and head-to-tail concatemers in most ofthe plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that-the kanamycinresistance marker is inherited as a single dominant gene.

A Polyethylene Glycol-Mediated Protoplast Transformation System for Production of Fertile Transgenic Rice Plants

“…Similar evidence for plasmid concatenation was previously reported in transgenic tobacco plants after protoplast transformation by electroporation (14). In tobacco, linear plasmid molecule appeared to form both head-to-head and head-totail concatemers.…”

Cotransformation and gene targeting in mouse embryonic stem cells.

“…5). Such concatemers of nonhomologously integrated DNA are common after calcium phosphate (20) and microinjection (8) transformation protocols and have been reported by other investigators after electroporation (2,3,23). In contrast, concatemers were rarely observed in earlier electroporation experiments by Toneguzzo et al (33) and by Boggs et al in our laboratory (4).…”

Presence of an SAR‐like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

“…Total DNAs from transformed and non-transformed BY-2 cells were isolated by the method of Mettler (1987) then puri®ed by equilibrium centrifugation in cesium chloride-ethidium bromide gradients (Sambrook et al, 1989) Cloning and sequencing of the junction regions Total DNA isolated from a transformant cell line (line 2±1) according to Mettler (1987) and puri®ed as described above, was ®rst digested with Stu I then fractionated by sucrose-density gradient centrifugation (Sambrook et al, 1989). The DNA fractions separated by electrophoresis were transferred to a nylon membrane and hybridized with the labeled npt II probe as described elsewhere (Iida et al, 1990a).…”