Introduction of resistance to kanamycin into the protoplasts from a pantothenate-requiring auxotrophic cell line of Datura innoxia P. Mill. via direct gene transfer

Abstract

The protoplasts from a pantothenate-requiring auxotrophic cell line of Datura innoxia were used as the host for plasmid DNA containing the neomycin phosphotransferase II (NPT-II) gene. DNA uptake was induced by incubating the protoplasts with an uptake- inducing solution containing 18% polyethylene glycol, 0.1 M calcium chloride, 0.05 M glycine, 70 mM sodium chloride and 2.5 mM potassium chloride following a heat shock at 45°C for 5 min. The selection of cell colonies resistant to kanamycin was performed on a medium supplemented with 40 mg/l kanamycin sulphate. Preculture of protoplasts after DNA treatment in the non-selective medium for 2 weeks was necessary for the recovery of transformants. The selected kanamycin-resistant cell lines were able to grow at more than 50% of the normal rates even in media containing 100 mg/l kanamycin. These cell lines were shown to express neomycin phosphotransferase enzyme activity in vitro. The presence of the NPT-II gene was confirmed by DNA analysis. The kanamycin-resistant cells remained auxotrophic after gene transfer. The presence of these two markers, auxotrophy and resistance to kanamycin, in the transformed cell lines renders them potential universal hybridizers for somatic cell fusion.