Organogenesis and plant selection by kanamycin of strawberry in vitro aiming at genetic transformation

Resumo

The success of genetic transformation of the strawberry (Fragaria x ananassa) is strongly influenced by the cultivar and depends on efficient regeneration and plant-transformed selection. In plant regeneration, efficient organogenesis depends on the concentrations of plant growth regulators that are present in the culture medium, and the response varies according to the explant type and cultivar. Similarly, in the selection of transformed plants, the sensitivity to selective agents induced by marker genes depends on the concentration of the selective substance in the regeneration medium and must be adjusted according to the chosen cultivar. The aim of this work was to test different combinations and concentrations of TDZ (Thidiazuron) and IBA (indolebutyric-acid) added to the base medium MS (MURASHIGE; SKOOG, 1962) for shoot regeneration of Camiño Real and Festival cultivars from leaf and petiole explants. In the case of ‘Camiño Real’, 1.0, 1.5 and 2.0 mg L-1 of TDZ combined with 0.1, 0.3 and 0.5 mg L-1 of IBA were tested. In the case of ‘Festival’, 1.0, 1.5 and 2.0 mg L-1 of TDZ combined with 0.1 and 0.3 mg L-1 of IBA were evaluated. Another aim was to define the lowest concentration of the selective agent Kanamycin (marker gene npt II) able to inhibit regeneration of the untransformed shoots. For this purpose, Kanamycin doses of 0, 2, 4, 6, 8 and 10 mg L-1 were added to the pre-established regeneration medium. In the petiole explant test, normal shoot regeneration did not occur; thus, only shoot regeneration and the sensitivity to Kanamycin from leaf explants were analyzed. For ‘Camiño Real’ the best shoot regeneration was established with a combination of 2.0 mg L-1 of TDZ + 0.1 mg L-1 of IBA. On the other hand, for ‘Festival’ the best shoot regeneration was established with a combination of 2.5 mg L-1 of TDZ + 0.1 mg L-1 of IBA. As far as Kanamycin sensitivity is concerned, total inhibition of the shoots was observed with 10 mg L-1 with both cultivars.