An efficient multiplex PCR assay for early detection of Agrobacterium tumefaciens in transgenic plant materials

The multiplex polymerase chain reaction (MPCR) is a variant of conventional PCR in which 2 or more target sequences are amplified by more than 1 pair of primers in a single reaction mixture. In this study, an efficient MPCR assay was established and optimized for the quick detection of Agrobacterium-derived plant transformants. The neomycin phosphotransferase II gene fragment (nptII; target gene) and the chromosomal virulence gene ChvA fragment of Agrobacterium tumefaciens (external reference gene) were amplified simultaneously with 2 pairs of corresponding primers. The MPCR results showed that 9 out of 10 kanamycin-resistant lines tested were transformed with the foreign genes. This optimized MPCR assay, rapid, sensitive, and reliable, can facilitate the identification of Agrobacterium-derived plant transformants at the early stage of selection.