GENE TARGETING AND INSTABILITY OF AGROBACTERIUM T-DNA LOCI IN THE PLANT GENOME

To develop a model system for studies of homologous recombination in p lants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia line s were generated harbouring a single target T-DNA containing the negat ive selective codA gene encoding cytosine deaminase (CD) and the beta- glucuronidase (GUS) gene. Subsequently, the target lines were transfor med with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For b oth Nicotiana species kanamycin-resistant lines were selected which ha d lost the CD gene and the GUS activity. One tobacco line was the resu lt of a precise gene targeting event. However, most other lines were s elected due to a chromosomal deletion of the target locus. The deletio n frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf proto plasts. T-DNA transfer was not required for induction of the deletions , indicating that the target loci were unstable. A few lines were obta ined in which the target locus had been deleted partially. Sequence an alysis of the junctions revealed deletion of DNA sequences between mic rohomologies. We conclude that T-DNAs, which are stable during plant d evelopment as well as in transmission to the offspring, may become uns table during propagation in callus tissue. The relationships between c allus culture, genetic instability and the process of T-DNA integratio n and deletion in the plant genome are discussed.