Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.
The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 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 detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.
Weld
R
R
Plant and Microbial Sciences Department, University of Canterbury, Christchurch, New Zealand. weldr@crop.cri.nz
Heinemann
J
J
Eady
C
C
D016923:Cell Death / Q000502:physiology; D002455:Cell Division; D004269:DNA, Bacterial / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation; D020868:Gene Silencing / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D014162:Transfection
D004269:DNA, Bacterial; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins