Agrobacterium T-DNA mutation causes the loss of GUS expression in transgenic tobacco

Abstract

A new freezer stock of an Agrobacterium tumefaciens clone of LBA4404/pBI121, designated 8999, was found to contain a mutation in the T-DNA region. GUS activity in Agrobacterium 8999 was reduced to levels in negative controls of LBA4404. Additionally, GUS activity in T1 seedlings from tobacco plants transformed with 8999 was reduced to that of untransformed plants. Southern and northern blotting showed that Agrobacterium clone 8999 transferred its T-DNA into the plant, that the correct sizes of 35S promoter and GUS coding region were integrated into the plant's genome in the correct orientation, but that no transcript was detectable after 24 h. Genomic DNA from a T1 seedling from 8999 transformation, digested with HpaII and MspI, indicated no methylation in the promoter region. We conclude from this data that Agrobacterium 8999 has a stable mutation that reduces expression at the mRNA level and is responsible for the lack of GUS expression in plants transformed with this Agrobacterium clone. Therefore, unselected genes within the T-DNA region may suffer mutations in Agrobacterium.