Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss

(T)he transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobac terium tumefaciens. Individual GFP-expressing protoplasts were isolated aft er 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost de tectable GFP activity during the first 4 weeks of culture. Multiple T-DNA c opies of the gfp gene were detected in 10 of 17 proto-calluses lacking visi ble GFP activity. The remaining 7 cell lines contained no gfp sequences. Ou r results confirm that transiently expressed T-DNAs can be lost during grow th of somatic cells and demonstrate that transiently expressing cells frequ ently integrate multiple T-DNAs that become silenced. In cells competent fo r DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integra tion.