Polymerase Chain Reaction Used for Monitoring multiple Gene Integration in <i>Agrobacterium</i>‐Mediated Transformation

Abstract

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 (NFT II) and β‐glucuronidase (GUS) in alfalfa ( Medicago saliva L.) transformed with Agrobacterium tumefaciens . We were able to distinguish between plants that contained only the NFT II gene and those that had both the NFT II and GUS genes. Of our putative transformants selected on kanamycin, 28% contained only the NFT 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.