Issue when expressing a recombinant protein under the control of p35S in Nicotiana tabacum BY-2 cells

Several recombinant proteins have been successfully produced in plants. This usually requires Agrobacterium -mediated cell transformation to deliver the T-DNA into the nucleus of plant cells. However, some genetic instability may threaten the integrity of the expression cassette during its delivery via A. tumefaciens , especially when the protein of interest is toxic to the bacteria. In particular, we found that a Tn3 transposon can be transferred from the pAL4404 Ti plasmid of A. tumefaciens LBA4404 into the expression cassette when using the widely adopted 35S promoter, thereby damaging T-DNA and preventing correct expression of the gene of interest in Nicotiana tabacum BY-2 suspension cells.