Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants |
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Authors: | Christian Brunold Susanne Krüger-Lebus Michael W Saul Samuel Wegmüller and Igo Potrykus |
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Institution: | (1) Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland;(2) Pflanzenphysiologisches Institut der Universität Bern, CH-4013 Bern, Switzerland;(3) Systematisch-Geobotanisches Institut der Universität Bern, CH-3013 Bern, Switzerland |
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Abstract: | Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR-K+) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR+K-SR+). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F1 plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR-K+) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner. |
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Keywords: | Protoplast fusion Nitrate reductase deficiency Kanamycin Nicotiana tabacum Nicotiana sylvestris |
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