Development of reliable, quick and cheap methods for screening transgene copy number and protein expression in transgenic plants

Transgenic plants require molecular analysis for screening and selection of high expressing target genes. In this study, the authors have developed rapid and high throughput methods for estimation of copy number and protein expression analysis. Transgene copy number, in transgenic plants greatly affects the expression level and genetic stability of the target gene, therefore estimation of transgene copy number is a key issue in genetically modified plant research. Traditionally, copy number has been determined by cumbersome and laborious blotting techniques, which requires large amount of genomic DNA and time. Agrobacterium-mediated transformation for the introduction of foreign genes into plants has been the favoured method. This transformation procedure generates predominantly low copy number insertion events, which simplifies assay development. The authors have developed a reliable and quick method that utilizes simple PCR assay to determine transgene copy level in plants. Using this assay, hundreds of samples can be analyzed per day in contrast to the low throughput encountered with traditional methods. This approach can also be successful for screening low copy and single copy events to other crops and transformation. A successful biofortification strategy requires a method to rapidly measure expressed protein concentration in food crops. The objective of this study was to develop a simple and reliable Enzyme-Linked Immunosorbent Assay (ELISA) using a polyclonal antibody to detect the overexpressed protein in transgenic rice seeds. The authors have used this technique to evaluate the expression levels and localization of various transgenic proteins-ferritin, OsNAS, hygromycin and Dehydrodipicolinate synthase and also have used an array of immunological methodology including in Situ Tissue Printing and Western Blot analysis. These techniques can be used in the future for various other proteins involved in drought tolerance and nutritional biofortification.