Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes

Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants withsilenced transgenes. To investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, Mexican lime [Citrus aurantifolia (Christm.) Swing.] plants were transformed with the p25 coat protein gene of Citrus tristeza virus (CTV) withor without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under nonselective conditions had silenced transgenes, and in all cases silencing affected all the three transgenes incorporated. These results indicate that the frequency of transgene silencing may be greatly underestimated when the rate of silencing is estimated from the number of regenerants obtained under selective conditions. To our knowledge, this is the first report in which the frequency of gene silencing after transformation has been quantified. When the integration pattern of T-DNA was analyzed in silenced and non-silenced lines, it was observed that inverted repeats as well as direct repeats and even single integrations were able to trigger gene silencing. Gene silencing has often been associated with the insertion of DNA sequences as inverted repeats. Interestingly, here, direct repeats and single-copy insertions were found in bothsile nced and non-silenced lines, suggesting that the presence of inverted-repeat T-DNAs and the subsequent formation of dsRNAs triggering gene silencing cannot account for all silencing events.