Expression of bacterial genes in plant cells.

Abstract

Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 ,jg/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.

Walnut: past and future of genetic improvement

“…Since identification of the Ti plasmid in Agrobacterium tumefaciens (Schell 1975) and development of the first genetically modified crop plant, an antibiotic-resistant tobacco plant (Fraley et al 1983), the public acceptance of genetically engineered plants has become a source of debate. Genetic engineering has been used successfully in J. regia since methods for somatic embryogenesis from immature cotyledons and plant regeneration were first developed (Tulecke and McGranahan 1985;McGranahan et al 1988McGranahan et al , 1990.…”

Anomalías en los comienzos de la transgénesis vegetal: intereses e interpretaciones en torno a las primeras plantas transgénicas

“…Mary-Dell Chilton was the first to obtain a transgenic plant, but there were also two other leading groups in the field of plant genetic engineering. In early 1983, at a conference on genetic engineering in plants, Schell and Van Montagu (of the University of Ghent), Rob Horsch and Fraley (of Monsanto) and Chilton (from Washington University) all announced similar results: all three groups had successfully expressed antibiotic genes in plant cells (Newton, 2010;Fraley et al, 1983;Herrera-Estrella et al, 1983;Chilton, 2001). All three groups had set out to find a method for transferring genes to plants, and they had succeeded.…”

Direct Gene Transfer to Plants

“…The APH(3')II activity migrated in non-denaturing polyacrylamide gels as two bands. The reason for this has not been defined, but it could obviously be due to post-translational modifications, proteolysis or association with other cellular protein(s) as previously found in animal cells (ColbereGarapin et al, 1981 , 1983bBevan et al, 1983;Fraley et al, 1983;Horsch et al, 1984;Murai et al, 1983). Experiments are now in progress to optimize the procedure for gene-mediated transformation of protoplasts.…”

“…In plants the only available method for genetic transformation is based on the ability of the soil bacterium Agrobacterium tumefaciens to transfer and to integrate part of its genetic material into genomic DNA (e.g., Bevan and Chilton, 1982). This complex natural transformation process has also been exploited for integrating functional foreign genes into the plant genome , 1983bBevan et al, 1983;Fraley et al, 1983;Horsch et al, 1984;Murai et al, 1983). Further, direct transformation of isolated Ti plasmid into protoplasts (Davey et al, 1980;Krens et al, 1982) has demonstrated that foreign DNA could be taken up by plant cells and integrated into the genome, but did not exclude the possibility that Ti plasmid-specific functions are essential for this transformation process.…”

“…glycoside phosphotransferase II [(APH3' )II] gene (Rothstein and Reznikoff, 1981). The protein encoded inactivates a related group of aminoglycoside antibiotics (neomycin, kanamycin, G-418) by phosphorylation and thereby confers resistance to them in bacteria (Rao and Rogers, 1979), fungi , mammalian cells (Colbere-Garapin et aL, 1981) and plant cells , 1983bBevan et al, 1983;Fraley et al, 1983). In order to obtain expression of the APH(3')II gene in higher eukaryotic cells, corresponding expression signals have to be provided.…”