Toxic effect of antibiotics in grapevine (<i>Vitis vinifera</i> 'Albariño') for embryo emergence and transgenic plant regeneration from embryogenic cell suspension
Regeneration of grapevine (Vitis vinifera L.) from embryogenic cultures after gene transfer is traditionally linked to a selection procedure using antibiotic containing media. The neomycin phosphotransferase II (npt-II) and hygromycin phosphotransferase (hpt) genes that confer resistance to the antibiotics kanamycin and hygromycin, respectively, have been the selectable marker genes most frequently used for selection of transgenic grapevines. In this work, the phytotoxic effects on embryo development and plant growth of these two antibiotics were examined in 'Albariño'. Embryogenic cell suspensions were evaluated based on a twostep strategy using untransformed and transformed tissues. The phytotoxic effect was significantly different at 20 mg∙L-1 (and higher) for kanamycin and at 5 mg∙L-1 (and higher) for hygromycin. Minimal killing concentrations of kanamycin and hygromycin for 'Albariño' cell suspensions were 50 and 12.5 mg∙L-1, respectively. Embryogenic cell suspensions were bombarded using the biolistic system with the construct pBI426, harboring the selectable npt-II gene, and incubated on kanamycin containing media to determine the best inhibitory concentration allowing embryo and shoot development of only transgenic events. Only 20 % of PCR-positive transgenic embryos and 20 % of plant regeneration resulted from embryos emerged on 30 mg∙L-1. However, 80 % of PCR-positive transgenic embryos but only 10 % of plant regeneration were obtained from embryos emerged on 40 mg∙L-1. The method described, based in untransformed and transformed plant material, could be used to determine the optimal antibiotic concentration for other V. vinifera cultivars for efficient selection and regeneration of transgenic events.
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