Plant Molecular Biology 45: 377–385, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
377
Transient GFP expression in Nicotiana plumbaginifolia suspension cells:
the role of gene silencing, cell death and T-DNA loss
Richard Weld
1,2,∗
, Jack Heinemann
1
and Colin Eady
2
1
Plant and Microbial Sciences Department, University of Canterbury, Private Bag480, Christchurch, New Zealand
(
∗
author for correspondence; e-mail: weldr@crop.cri.nz);
2
New Zealand Institute for Crop and Food Research,
Private Bag, Christchurch, New Zealand
Received 30 November 1999; accepted in revised form 30 July 2000
Key words: Agrobacterium tumefaciens, gene silencing, GFP, tobacco, transient expression
Abstract
The transient nature of T-DNA expression was studiedwith a gfp reporter gene transferred to Nicotiana plumbagini-
folia 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.
Introduction
Procedures based on the natural transformation sys-
tem of Agrobacterium are widely used to transfer
genes into plant cells. After co-cultivation of plant
cells with Agrobacterium, T-DNA expression and the
frequency of expressing plant cells increases rapidly,
peaks within a few days and then declines over subse-
quent weeks (Janssen and Gardner, 1989; Mozo and
Hooykaas, 1992; Narasimhulu, 1996; Yoshioka et al.,
1996). This temporal profile of transgene expression
led to the conclusion that there was transcription from
unstable extrachromosomal copies of the T-DNA that
were not maintained in the plant cell (Janssen and
Gardner, 1989; Yoshioka et al., 1996). Such a model
of transient expression is supported by the observa-
tion that ecotypes of Arabidopsis that integrate T-DNA
inefficiently are relatively efficient at expressing T-
DNA transiently (Sonti et al., 1995; Nam et al., 1997,
1998). Further, co-transformation of tobacco with T-
DNA carrying the cre gene and T-DNA carrying lox
sites can result in lox recombinants lacking the cre
vector (Vergunst and Hooykaas, 1998). As cre expres-
sion is probably required for lox recombination, cre
was probably transiently expressed and subsequently
lost from the plant cells. Finally, tobacco shoots re-
generated without selection from co-cultivated tissue
have been observed to rarely contain integrated but un-
expressed T-DNA, suggesting that gene silencing does
not play a significant role in transient expression (De
Buck et al., 1998). As a consequence of the paradigm
of transient expression, it has become widely assumed
that T-DNA integration into the plant genome is a
major factor limiting the frequency of ‘stable’ trans-
formation events upon Agrobacterium-mediated gene
transfer (Sonti et al., 1995; Narasimhulu et al., 1996;
De Buck et al., 1998).
Transient expression is often used in the optimiza-
tion of transformationprotocolsas an indicator of gene
transfer and expression efficiency (Liu et al., 1992; Li
et al., 1992; Gallo-Meagher and Irvine, 1993; Eady
et al., 1996; Jain et al., 1996; Kapila et al., 1997;
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