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

Abstract

Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants with silenced 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) with or without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under non-selective 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 both silenced 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.

Consistent transcriptional silencing of 35S‐driven transgenes in gentian

“…In addition, transgene silencing might potentially affect the screening efficiency of transformants. This assumption is supported by the observation that approximately 30% of transgenic Mexican limes showed transgene silencing after non-selective screening, whereas no silencing was found after selection culture (Domínguez et al, 2002). This result suggests that low transformation frequencies in some plant species might be attributed to the frequent occurrence of transgene silencing in their transformation process.…”

“…In addition, nuclear run-on transcription analysis did not detect any bar gene transcripts in the silenced plant lines ( Figure S6), indicating that gentian transgene silencing might be associated not with PTGS, but rather with TGS. As a certain proportion of transgenic Mexican limes regener-ated under non-selective conditions showed transgene silencing associated with methylation (Domínguez et al, 2002), it is possible that other plant species from which it is difficult to obtain transformants might share the same genesilencing trait as gentian. Thus information on the gentian transgene methylation machinery might provide a clue for the production of functional transgenic plants.…”

Citrus tristeza virus: a pathogen that changed the course of the citrus industry

“…The same strategy has been used to transform other Citrus hosts, but results from challenge experiments were unclear or not reported (Febres et al ., 2003; Ghorbel et al ., 2000; Gutiérrez et al ., 1997). Lime and grapefruit plants transformed with untranslatable versions of the CP gene to achieve RNA‐mediated resistance did not prevent CTV infection and the protection afforded was rapidly overcome by the challenging virus (Domínguez et al ., 2002a,b; Febres et al ., 2003; Herron et al ., 2002). For the same purpose, constructs derived from the CTV 3′‐UTR were used to transform sweet orange protoplasts and grapefruit plants but conclusive results on protection at the whole plant level were not reported (Febres et al ., 2003; Olivares‐Fuster et al ., 2003).…”

Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T‐DNA integrations

“…In order to investigate genome‐wide apparent non‐randomness of T‐DNA integrations, we have taken advantage of several recently developed transformation systems that do not depend on activity of selectable markers (Dominguez et al. , 2002; Permingeat et al.…”

“…These systems use polymerase chain reaction (PCR) to identify transformants regenerating in the absence of selection and are therefore free from selection bias. It is interesting that some of these selection‐free systems have reported a high occurrence of transcriptional gene silencing (TGS) (Dominguez et al. , 2002).…”