High-resolution structural analysis of biolistic transgene integration into the genome of wheat

Transformation of plant genomes by biolistic methods has become routine ove r the past decade. However. relatively little is known about how transgenes are physically integrated into the host genome. Using a high-resolution ph ysical mapping technique, fluorescence in situ hybridization on extended DN A fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed to determine the structural arrangement of stably inherited transgenes in h ost-plant chromosomes. Twelve transgenic lines were transformed with a sing le plasmid and one line was co-transformed with two separate plasmids, whic h co-segregated genetically. Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integrati on; Type II, large tandem integrations interspersed with unknown DNA: and T ype III, small insertions. possibly interspersed with unknown DNA. Metaphas e FISH showed that the integration of transgenes was in both hetero- and eu chromatic. as well as proximal. interstitial and distal. regions of the chr omosomes. In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transg ene expression. The integration of the transgenes was not associated with d etectable chromosomal rearrangements.