An efficient method for the physical mapping of transgenes in barley usingin situ hybridization

The genetic transformation of crops by particle bombardment and Agrobacteri um tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of thi s process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This stu dy describes a new approach, which provides efficient control of probe leng th and labelling, both of which play an important role in in situ hybridiza tion of transgenes. The approach is based on reducing the size of the plasm id prior to labelling by nick translation, rather than using the whole or l inearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which y ielded optimal hybridization, minimal fluorescent background, and accurate physical location of the transgene.