POLYMERASE CHAIN-REACTION USED FOR MONITORING MULTIPLE GENE INTEGRATION IN AGROBACTERIUM-MEDIATED TRANSFORMATION

Characterizing plants following Agrobacterium-mediated transformation has become an important consideration in the creation of transgenic plants. As an alternative to Southern blot analysis, polymerase chain reaction (PCR) was used to screen for the simultaneous integration of two foreign genes for neomycin phosphotransferase II (NPT II) and beta-glucuronidase (GUS) in alfalfa (Medicago sativa L.) transformed with Agrobacterium tumefaciens. We were able to distinguish between plants that contained only the NPT II gene and those that had both the NPT II and GUS genes. Of our putative transformants selected on kanamycin, 28% contained only the NPT II gene and apparently had lost the GUS gene during the transformation process. Requiring only nanogram amounts of DNA, PCR provided a rapid method to determine which genes had been integrated to our putative transformants much earlier in the plant regenerative process than was feasible by Southern analysis.