Quantitative real-time PCR assay for determining transgene copy number in transformed plants

The development of transgenic events call be limited by many factors. These include expression levels, insert stability and inheritance, and the ident ification of simple insertion events. All of the factors can be related kto tire copy number of the transgene. Traditionally copy number has been dete rmined by laborious blotting techniques. We have developed an alternative a pproach that utilizes the fluorogenic 5' nuclease (TaqMan (R)) assay to qua ntitatively deter mine transgene copy level in plants. Using this assay, hu ndreds of samples colt be analyzed per day in contrast to the low through-p ut encountered with traditional methods. To develop the TaqMan copy number assay, we chose to utilize our highly efficient Agrobacterium-mediated tran sformation system of maize. This transformation procedure generates predomi nantly low copy number insertion events, which simplified assay development . We have also successful applied this assay to other crops and transformat ion systems.