1062-3604/04/3503- © 2004
Russian Journal of Developmental Biology, Vol. 35, No. 3, 2004, pp. 174–181. Translated from Ontogenez, Vol. 35, No. 3, 2004, pp. 220–228.
Original Russian Text Copyright © 2004 by Ogarkova, Tomilov, Tomilova, Tarasov.
By the end of 2000, the complete nucleotide
sequence of the ﬁrst species of higher plants,
, was deciphered (http//www.arabidop-
sis.org). Although the genetics of
actively developing for many years, the functions of
most of its genes are not yet established. As a result, the
problem of identiﬁcation of the
already determined nucleotide sequences was has
brought come to the forefront.
It is evident that, to establish a relationship between
the a character and a genetic determinant, which is
specifying this character, it is necessary to change
somehow the expression of this determinant. Although
the methods of suppression of gene activities by anti-
sense RNAs have recently been elaborated (Bouchez
and Hofte, 1998; Tissier
, 1999), the traditional
method of mutagenesis still remains the main method
of gene function identiﬁcation.
The knowledge of the complete nucleotide sequence
radically changes the technology of studying the struc-
tural–functional organization of the genome. Speciﬁ-
cally, in studies aimed at identiﬁcation of gene func-
tions, this circumstance makes it possible to use the
gene-directed mutagenesis related to substitution of the
an intact gene by its mutant copy via homologous
recombination. This method is widely used for many
model organisms, such as microorganisms and mam-
mals (Bouchez and Hofte, 1998), but its application to
higher plants is complicated, since homologous recom-
bination proceeds in them with a low efﬁciency (Puchta
and Hohn, 1996).
Advances in the studies of functional organization
genome are related to the develop-
ment of insertional mutagenesis (Azpiroz-Leehan and
Feldmann, 1997; Bouchez and Hofte, 1998; Meissner
, 1999), and of special signiﬁcance is the produc-
tion of collections of mutants with the help of inser-
tional mutagenesis. Such collections have already
obtained in some laboratories (Krysan
, 1999; Speulman
, 1999; Tissier
, 1999; Ogarkova
In order to characterize the phenotype of insertional
mutants with hereditary morphological
changes, the classiﬁcation ﬁrst proposed by Feldmann
(1991) is currently used. It includes seven phenotypic
classes, including the class “lethal germlings,” which
accounts to 3–5% of all obtained insertional mutants.
The mutants of this class do not form the ﬁrst pair of
true leaves and die at the age of up to two weeks.
The aim of this work was to save insertional reces-
sive lethal mutants of this phenotype for identiﬁcation
of the mutant character and gene, insertion into which
determines a given phenotype.
MATERIALS AND METHODS
. The collection of insertional mor-
phological mutants was used as the genetic material,
which was obtained using the method of agrobacterial
transformation of germinating seeds (Feldmann and
Marks, 1987; modiﬁed by Ogarkova
, 1997; Tom-
, 1999) with the help of binary vector systems
, 1997) and pPCVRN4 (Koncz
, 1994) and of supervirulent strain
Media and conditions of cultivation.
germinated in sterile conditions on a vitamin- and
sucrose-free agarized (0.8%) medium of Kvitko (1960)
containing marker antibiotics: 50 mg/l kanamycin in
GENETICS OF DEVELOPMENT
Saving Insertional Recessive
Lethal Mutants of
O. A. Ogarkova, A. A. Tomilov, N. B. Tomilova, and V. A. Tarasov
Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina 3, Moscow, 119991 Russia
Received August 7, 2003; in ﬁnal form, October 20, 2003
—A group of 13 recessive lethal mutants was selected on the basis of the collection of
transgenic plants with insertions of T-DNA vector plasmid pLD3 or pPCVRN4, which was produced
by agrobacterial transformation of germinating seeds. The use of media containing exogenous hormones made
it possible to compensate the lethal effect, identify phenotypes, and characterize six lines of recessive lethal ger-
mlings using genetic and molecular-genetic methods.
: insertional mutagenesis, exogenous hormones, saving lethal germlings.