Developmental timing of transgene expression is dosage dependent

Expression of a β ‐glucuronidase (GUS) gene ( uidA ) driven by the cauliflower mosaic virus 35S promoter was studied during development in progeny from a primary transformant of tobacco carrying 3 independently segregating inserts. In the R1S2‐generation, plants could be grouped to 4 GUS expression phenotypes, showing either continuous GUS activity [S(‐) plant] or silencing of GUS after 13, 5 or 2 weeks [S(13), S(5) and S(2) plants, respectively]. In S(13), S(5) and S(2) plants, GUS activity was reduced 7‐, 17‐ and 50‐fold, respectively, as compared to the S(‐) plant. Southern blot and segregation analysis showed that S(2) plants contained all 3 inserts of which at least 1 was homozygous. All S(5) plants contained 2 inserts, 1 of which was homozygous and the other hemizygous. The S(13) plant contained 2 hemizygous inserts, while the S(‐) plant contained 1 hemizygous insert. Analysis of the R1S3‐generation showed that the timing and degree of silencing was directly linked to copy number and that each of the 3 inserts seemed to contribute equally to gene silencing. The physically linked neomycin phosphotransferase II gene driven by the weak nopaline synthase promoter was also subject to dosage‐dependent gene but this occurred later and was less pronounced.