Assessment of CaMV-mediated gene silencing and integration of CaMV into GM plants with a 35S RNA promoter.

Four GM plant species (Arabidopsis thaliana, Brassica napus, Nicotiana benthamiana and N. tabacum), each expressing the gene encoding the jellyfish green fluorescent protein (GFP) regulated by the cauliflower mosaic virus (CaMV) 35S RNA promoter, were assessed for the extent of transgene silencing and viral genome integration following infection by CaMV. The first three species are systemic hosts of CaMV, while N. tabacum is only a local host for a few strains of CaMV. A generalized systemic silencing of the GFP transgene was not observed in a total of 100 plants of each species infected with CaMV, although some localized loss of GFP was observed in CaMV-infected N. benthamiana leaves, and some loss of fluorescence was observed in older leaves of uninfected as well as infected plants. Progeny seedlings obtained from the above infected plants also did not exhibit transgene silencing showing that virus infection did not affect the stability of the transgene. These progeny plants also did not show signs of virus infection, indicating that the presence of the CaMV 35S RNA promoter sequences in the plant genome did not potentiate seed transmission of the virus. Integration of infective CaMV into the CaMV 35S RNA promoter could not be detected in 944 samples taken from leaves of the above infected plant species or in 2912 samples taken from progeny seedlings. Based on a detection limit of one copy per 4000 haploid genomes, we conclude that if integration of virus does occur into the CaMV 35S RNA promoter, then it occurs at such a low frequency as to be insignificant.