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Genetic transformation of apple ( Malus x domestica ) without use of a selectable marker gene

Malnoy, Mickael ; Boresjza-Wysocka, Ewa ; Norelli, John ; Flaishman, Moshe ; Gidoni, David ; Aldwinckle, Herb

In: Tree Genetics & Genomes, 2010, vol. 6, no. 3, p. 423-433

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    Title
    • Genetic transformation of apple ( Malus x domestica ) without use of a selectable marker gene
    Author
    • Malnoy, Mickael. IASMA Research Center, E. Mach Foundation, Via E. Mach 1, 38010, San Michele all'Adige, Trento, Italy
    • Boresjza-Wysocka, Ewa. Department of Plant Pathology, Cornell University, 14456, Geneva, NY, USA
    • Norelli, John. USDA-ARS Appalachian Fruit Research Station, 25430, Kearneysville, WV, USA
    • Flaishman, Moshe. The Department of Fruit Tree Sciences, Institute of Horticulture, ARO, The Volcani Center, 50250, Bet Dagan, Israel
    • Gidoni, David. The Department of Fruit Tree Sciences, Institute of Horticulture, ARO, The Volcani Center, 50250, Bet Dagan, Israel
    • Aldwinckle, Herb. Department of Plant Pathology, Cornell University, 14456, Geneva, NY, USA
    Document Type
    • Postprint
    OAI-PMH Identifier
    • oai:doc.rero.ch:314053
    Summary
    Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype "M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0-25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, "Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13%