ISSN 1022-7954, Russian Journal of Genetics, 2009, Vol. 45, No. 5, pp. 546–551. © Pleiades Publishing, Inc., 2009.
Original Russian Text © V.A. Tarasov, G.V. Pogorelko, O.A. Ogarkova, 2009, published in Genetika, 2009, Vol. 45, No. 5, pp. 624–630.
The main method to determine the functions of genes in
a model object of higher plants
insertional mutagenesis. Abundant collections of insertional
mutants have been obtained at present with the use of this
method. Their seeds are stored in a number of collection
centers, such as ABRC (http://www.arabidopsis.org/abrc/),
NASC (http://nasc.nott.aac.uk/), SALK (http://signal.
salk.edu/), GABI (http://www.gabi-kat.de/), and others.
Most of the available collections of insertional mutants
contain lines carrying so-called loss-of-function (null)
mutations that completely disturb the function of a mutant
gene. In this connection, an important task in elucidating
the functional signiﬁcance of genes of higher plants is to
extend the spectrum of mutations (in particular, to obtain
collections of mutants caused by superexpression of genes).
To obtain mutants with superexpression of genes, spe-
cial systems of vectors were created in which T-DNA con-
tains an enhancer [1, 2]. Two types of mutations can occur
when an enhancer-containing insertion is integrated in the
plant genome: ﬁrstly, these are null mutations associated
with inactivation of genes with an insertion integrated in
their nucleotide sequence; secondly, a mutant phenotype
can be caused by superexpression of genes containing no
A system of vectors was created by us earlier to induce
and select mutants associated with the presence of an
enhancer in the structure of T-DNA . In this work, the
efﬁciency of the system of generation and selection of inser-
tional mutants with superexpression of genes was studied.
MATERIALS AND METHODS
, Columbia ecotype, were used.
Sterilization of seeds, media, and cultivation condi-
tions were described in detail previously [3, 4].
plants were grown in sterile
conditions using Kvitko’s medium  or enriched
Milieu Arabidopsis medium (DUCHEFA, Netherlands)
up to a concentration of
1 M and with bactoagar (DUCHEFA, Netherlands)
up to 1% concentration. Acidity was brought to pH 5.8
plants were grown in standard
soil (pH 6.0) with 16 h ﬂuorescence illumination in a
and 60% humidity. Phillips
Biolux (spectrum 930 or 945) and Osram Fluora (pink
spectrum) lamps were used.
Bacterial strains and plasmids used to create a sys-
tem of vectors for generating and selecting insertional
mutants determined by superexpression of genes were
described previously .
C58 strain was trans-
formed with plasmids pEnLox and pCre by direct trans-
fer of plasmid DNA using the freezing–thawing
DNA isolation, puriﬁcation, hybridization, molecu-
lar cloning, sequencing, and PCR were performed
according to conventional procedures  with the use
of commercial kits (Promega, Fermentas, Invitrogen).
Agrobacterium-mediated transformation of
was carried out by the ﬂoral dip method (inﬁltration of
immature buds) . In the case of the pEnLox vector,
after cocultivation with agrobacteria, the plants were
grown to the stage of mature seeds (T1 generation). The
seeds (T2 generation) were sown on soil, after which
they were sprayed six times every 2–3 days with a
water solution of the herbicide ammonium glufosinate,
known as Basta (250 mg/l). Mature seeds (T3 genera-
tion) were collected from Basta-resistant plants. In the
case of the pCre vector, transformants were selected in
the T2 generation on a selective medium with the anti-
biotic hygromycin (30
Genes Whose Expression
Determines Changes in the Shoot Structure
V. A. Tarasov, G. V. Pogorelko, and O. A. Ogarkova
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia;
Received January 30, 2008
—A system was created to obtain and select
genes whose superexpression
causes development of a mutant phenotype. Three morphological mutants (two with a markedly retarded
growth and one with a fasciated stem) associated with the superexpression of genes
were generated with the use of this system. Localization, structure, and a possible functional orga-
nization of these genes were determined.