Plant Molecular Biology 50: 551–562, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Seedling lethality in Nicotiana plumbaginifolia conferred by Ds
transposable element insertion into a plant-speciﬁc gene
Amel Majira, Monique Domin, Olivier Grandjean, Krystyna Gofron and Nicole Houba-H
Laboratoire de Biologie Cellulaire INRA, Route de St-Cyr, 78026 Versailles Cedex France (
author for correspon-
dence; e-mail firstname.lastname@example.org)
Received 12 September 2001; accepted in revised form 12 March 2002
Key words: Ds, elongation, seedling lethal, transposon tagging
A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize
Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with
two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phe-
notypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells,
that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions.
Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Comple-
mentation of the mutant phenotype with the cDNA conﬁrmed that the transposon had caused the mutation. The Ds
element was inserted into the ﬁrst exon of the open reading frame and the homozygous mutant lacked detectable
transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein
found in several plant genomes but apparently missing from animal and fungal genomes; the protein is highly
conserved and has a potential plastid targeting motif.
Insertional mutagenesis using endogenous transpos-
able elements has been very powerful in correlating
mutant phenotypes with speciﬁc disrupted genes in
maize and snapdragon (Gierl and Saedler, 1992; Wal-
bot, 1992, for reviews). Insertional mutagenesis was
greatly expanded when maize transposable elements
were engineered to transpose in heterologous hosts.
Presently huge programs seek saturation mutagen-
esis with maize transposons, namely MuDR/Mu in
maize and Ac/Ds or Spm/dSpm in Arabidopsis thaliana
(Parinov and Sundaresan, 2000; Walbot, 2000, for
reviews). Host gene sequences ﬂanking transposon
insertions (FST) can be catalogued to allow sequence-
based searches for insertion mutations of interest.
Despite the beneﬁts of transposon tagging for re-
verse genetics, widespread adoption of this method is
limited by transformation methods to introduce func-
tional transposons into new hosts. Many crop plants,
particularly the grasses, would beneﬁt from a more
general method for transposon introduction (Greco
et al., 2001).
Nicotiana plumbaginifolia is a true diploid species.
Bourgin et al. (1979) established a haploid line from
pollen culture and showed that plants could be eas-
ily regenerated from mesophyll protoplasts. Success-
ful regeneration could also be achieved with proto-
plasts prepared from haploid cell suspension cultures
(Barﬁeld et al., 1985). Classical mutagenesis on hap-
loid protoplasts followed by selection for auxotrophy
or resistance to toxic compounds at the protoplast-
derived cell level generated metabolic mutants useful
in biochemical studies (Negrutiu, 1990, for review).
N. plumbaginifolia remains a model species for phys-
iological and gene expression research (El Malki and
Jacobs, 2001; Fraisier et al., 2001). Suitable inducing
treatments on N. plumbaginifolia cell cultures are even
amenable to gene isolation (Jasinski et al., 2001).
In Nicotiana plumbaginifolia few endogenous
transposons have been described and they are mainly
retrotransposons (Meyer et al., 1994 ; Leprince et al.,