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

Abstract

SummarySeveral recent investigations of T-DNA integration sites in Arabidopsis thaliana have reported 'cold spots' of integration, especially near centromeric regions. These observations have contributed to the ongoing debate over whether T-DNA integration is random or occurs preferentially in transcriptionally active regions. When transgenic plants are identified by selecting or screening for transgenic activity, transformants with integrations into genomic regions that suppress transcription, such as heterochromatin, may not be identified. This phenomenon, which we call selection bias, may explain the perceived non-random distribution of T-DNA integration in previous studies. In order to investigate this possibility, we have characterized the sites of T-DNA integration in the genomes of transgenic plants identified by pooled polymerase chain reaction (PCR), a procedure that does not require expression of the transgene, and is therefore free of selection bias. Over 100 transgenic Arabidopsis plants were identified by PCR and compared with kanamycin-selected transformants from the same T 1 seed pool. A higher perceived transformation efficiency and a higher frequency of transgene silencing were observed in the PCR-identified lines. Together, the data suggest approximately 30% of transformation events may result in non-expressing transgenes that would preclude identification by selection. Genomic integration sites in PCR-identified lines were compared with those in existing T-DNA integration databases. In PCR-identified lines with silenced transgenes, the integration sites mapped to regions significantly underrepresented by T-DNA integrations in studies where transformants were identified by selection. The data presented here suggest that selection bias can account for at least some of the observed non-random integration of T-DNA into the Arabidopsis genome.

A Common Histone Modification Code on C4 Genes in Maize and Its Conservation in Sorghum and Setaria italica

“…Promoter-deletion studies were instrumental in identifying such functional elements. However, these deletion studies require the use of transgenes that randomly integrate into plant genomes (Francis and Spiker, 2005;Kim et al, 2007). Epigenetic traits such as NO and histone modification can be strongly affected by the transgene integration site (Yan and Boyd, 2006;Yamasaki et al, 2011;Yin et al, 2012).…”

Expression of Bacillus thuringiensis insecticidal protein gene in transgenic oil palm

“…Many reports have shown that transgenes may be present in its new environment but were not expressed or expressed at a very low level making it undetectable. Failure for transgene to express may be due to mutations in the transgenes (Tinland, 1996), post-transcriptional gene silencing (Mitsuhara et al 2002;Szittya et al 2003), or chromatin-related transgene silencing (Francis and Spiker, 2005). In this study, although PCR analyses could easily detect the presence of CryIA(b) in the putative transformed plants, but attempts to detect its expression and functionality could not be easily carried out.…”

“…Evaluation of transgene expression in T 0 transgenic plants has always been difficult, especially when the transgenes undergoes various modifications in its new environment (Stam et al 1998;Francis and Spiker, 2005). Many reports have shown that transgenes may be present in its new environment but were not expressed or expressed at a very low level making it undetectable.…”

Genetic engineering of cotton with a novel cry2AX1 gene to impart insect resistance against Helicoverpa armigera

“…A wide range of Bt protein expression in transgenic plants with the same genetic background and gene construct were reported by several earlier studies (Maqbool et al 2001, Breitler et al 2000, Ramesh et al 2004, K Dhivya et al Meiyalaghan et al 2006). We reason that the variation in the level of transgene expression or lack of expression may be due to mutations in the transgene, truncation of T-DNA during integration, post transcriptional gene silencing, or transcriptional gene silencing (integration of T-DNA into genomic regions such as the heterochromatin that repress transgene expression) (Francis and Spiker 2005). Inactivation of the transgene is often shown to be accompanied by an increase in DNA methylation (Amasino et al 1984).…”