Rapid PCR-based determination of transgene copy number in rice

We present a simple, rapid, and low-cost method to determine transgene copy number in rice. More than 100 first- and second-generation transgenic rice plants were tested. The plasmid (pRCopy) used for rice transformation contains the specific gene of interest and a partially deleted cytochrome c gene (cyc), a single-copy gene in rice. A 132-bp segment of the cloned ricecyc was shortened to 108 bp by deleting a 24-bp internal fragment. After PCR amplification of the genomic DNA from transgenic rice harboring pRCopy, the 2 expected bands were found. The 121-bp band corresponds to the endogenouscyc; the 97-bp band comes from the integrated pRCopy. Clear distinctions can be made between single and multiple copies of the transgene by comparing band densities.