Stable transformation of sugarbeet protoplasts by electroporation

Conditions were optimized for the culture, antibiotic selection and stable transformation by electroporation of suspension culture protoplasts of sugarbeet,Beta vulgaris L.. Highest plating efficiencies (up to 65% at day 21) were obtained if protoplasts were cultured in PGO salts (de Greef and Jacobs, 1979) supplemented with 0.1 mg/1 2,4-D, 0.01 mg/l BAP and 9% mannitol, and in 0.6% agarose rather than in liquid medium. Sensitivity to kanamycin also depended on whether protoplasts were cultured in liquid or agarose medium. Stable transformation of protoplast-derived colonies, as determined by resistance to kanamycin and Southern blot analysis, was achieved by electroporation using both rectangular and exponentially-decaying pulses.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - npt-II neomycin phosphotransferase - EDTA ethylenediaminetetracetic acid - SDS sodium dodecyl sulphate