THE USE OF COMBINED FISH GISH IN CONJUNCTION WITH DAPI COUNTERSTAINING TO IDENTIFY CHROMOSOMES CONTAINING TRANSGENE INSERTS IN AMPHIDIPLOIDTOBACCO/

We have used combined fluorescent and genomic in situ hybridization (F ISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterst aining to determine simultaneously the chromosome integration site and subgenomic allocation of a transgene in-sert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4x = 48). The procedure provides sufficient i nformation on physical markers to identify at least 20 out of 24 chrom osome pairs of two tobacco cultivars commonly used in studies on trans gene expression and silencing (cv. Petit Havana SR1 and cv. Gaterslebe n). The chromosomes can be distinguished on the basis of diploid paren tal ancestry, size, morphology, the presence of rDNA loci and/or inter genomic exchanges, and the DAPI banding pattern, which is shown here f or the first time for N. tabacum. From a single ISH experiment, it sho uld now be possible in most cases to identify a tobacco chromosome car rying a transgene insert, thus permitting systematic studies of how th e chromosome location of transgenes influences expression levels.