Real Time quantitative PCR Analysis of Transgenic Maize Plants Produced by Agrobacterium-mediated Transformation and Particle Bombardment

Abstract

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene. Thus, making estimation of transgene copy number and determination of the expression levels an important area of genetically modified crop research. The analysis of transgenic plants can be as important as the transformation process itself. After producing the primary transformants, it is very important to decide which plants contain the transgene and in how many copies[7]. While multiple copies are useful for over-expression experiments, single or low copy transformation events are preferred for most applications because they are stable over several generation of subsequent breeding[24]. The analysis of transgene integration has been recently relied on PCR-based methods, which require only small amounts of plant material and are easily automated for high-throughput quantita tion . Conventional PCR is commonly used for screening large numbers of putative transformants for transgenic sequences[23] and as an alternative to marker selection for segregation analysis[18]. However, conventional PCR analysis is plagued by false positives from contamination because a band with the size of the desired product is counted as signal, regardless of the intensity. Transgene copy number has traditionally been estimated by Southern analysis, although recently other methods, including comparative genomic hybridization, fluorescence in situ hybridization, multiplex amplifiable probe hybridization and microarray have been applied to copy number estimation. Unfortunately, all of these methods are laborious and time-consuming, require considerable amount of DNA from fresh or frozen samples and often involve the use of hazardous radioisotopes[34]. Real time PCR (ABI Prism 7700 Sequence Detection System) has been recently established as a rapid and sensitive technique for precise quantitation[13]. With real-time PCR as a quantification tool, it is possible to rapidly analyze large numbers of putative transformants from high-throughput transformation procedures[7]. Real-time PCR was initially used in plant research as a highly specific and sensitive detection