Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

Genomic DNA blot hybridization is traditionally used to demonstrate that, v ia genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomi c DNA blot hybridization is pushed towards its limits, because a considerab le quantity of DNA is needed to obtain enough genome copies for a clear hyb ridization pattern. Furthermore, genomic DNA blot hybridization is a time-c onsuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DN A borders does not have these drawbacks and seems to be an adequate alterna tive to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA w as integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The re sults showed that T-DNA integration took place and gave insight in the numb er of T-DNA copies present. Comparison of AL-PCR and previously obtained ge nomic DNA blot hybridization results pointed towards complex T-DNA integrat ion patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that up on T-DNA integration a 66 bp genomic sequence was deleted, and no filler DN A was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T- DNA integration.