Quantitative analysis of transgenes in cassava plants using real-time PCR technology

Abstract

To speed up the molecular analysis of cassava transgenic plants, we developed real-time polymerase chain reaction (PCR)-based methods that could be implemented as a tool in the primary scrutiny of putative transgenic plants. We tested for the presence of transgenes, estimated copy number, and quantified messenger RNA (mRNA) levels of genes introduced through Agrobacterium. Copy numbers for the genes ß-glucuronidase and hygromycin phosphortransferase were estimated in 15 transgenic lines. Most lines contained one or two copies of each gene; in some, the copy number was different for the two genes, suggesting rearrangements of the transferred DNA. Six of the 15 lines were analyzed by Southern blot. The copy number so estimated was concordant in most cases. Although real-time PCR was efficient for classifying transgenic lines with one or more transgenes inserted, for conclusive analysis of gene copy number, i.e., in a potential breeding line, the Southern blot may still be required. The transcript levels from both genes were determined in eight lines. High, medium, and low levels of mRNA expression were detected. No direct relationship between copy number and expression level of transgenes was obvious, suggesting that factors like position effects or DNA rearrangements led to differential expression. Quantitative mRNA expression data for the ß-glucuronidase gene agreed with results from histochemical staining. With real-time PCR we could detect high levels of transgene expression in 3-y-old cassava plants maintained and propagated as clones in the greenhouse. This is the first time that real-time PCR is reported to be used for transgene analysis in cassava