Browsing by Subject "'Piel de Sapo'"
  • Institution Publication
    Selection of Cucumis melo L. subsp. melo 'Piel de Sapo' lines with high efficiency in Agrobacterium-mediated genetic transformation
    ( 2012-03-31)
    Menéndez E.
    ;
    Marfa V.
    ;
    Claveria E.
    ;
    Martínez I.
    ;
    Pujol M.
    ;
    Dolcet-Sanjuan R.
    There are important hindrances for setting up a routine melon transformation protocol, including the strong effect of genotype and the high incidence of polyploid regenerants. Genetic transformation efficiencies for melon lines of Cucumis melo subsp. melo 'Piel de Sapo' inodorus type (e.g., 'T111'), are lower than those obtained with the 'Védrantais' line or the accession C. melo subsp. agrestis 'PI 161375' (Castelblanque et al., 2008). Four near isogenic lines (NILs), SC 6-6, SC 7-2, SC 7-4, and SC 8-4, selected for their higher regeneration capacity from a population of 32 NILs, and the 'T111' line were used to determine their genetic transformation efficiency via A. tumefaciens. These NILs were developed from a doubled haploid line (DHL) population derived from the cross between the Korean accession 'PI 161375' and the Spanish 'T111'. Transgenic melon plants were produced from 'T111' and three NILs, using cotyledon explants inoculated with an A. tumefaciens strain harboring a binary vector containing genes encoding β-glucuronidase activity and conferring tolerance to glyphosate, as selection marker. Cotyledonary explants were cultured on the regeneration medium supplemented with 4 mg L-1 Basta herbicide. Putative transformed plantlets were tested for gene insertion 3-4 months after inoculation. Transformants were selected on the basis of glyphosate tolerance and GUS expression on leaves and roots. Following such protocol, the transformation efficiency has increased from a 3.3%, for the 'Piel de Sapo' line 'T111' to 23.3% for two of the selected NILs. Out of 105 transgenic plants produced, 11% were diploid and the rest were tetraploid. Higher ploidy levels usually result in sterility, and failure to perpetuate the transgenic line. Another source of explants is under study to facilitate the recovery of diploid transgenic plants.