1062-3604/05/3603- © 2005 Pleiades Publishing, Inc.
Russian Journal of Developmental Biology, Vol. 36, No. 3, 2005, pp. 181–183. Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 222–224.
Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Tarasov.
Although the genetics of
been developed for several tens of years and the
, race Columbia was fully
sequenced in 2000, the functions of only a small part of
its genes have been identiﬁed. Thus, the problem of
identiﬁcation of the
genes was brought to
Advances in studying the functional organization of
genome have been largely related to the
development of the methodology of insertion mutagen-
esis and of the collections of insertion mutants. Such
collections have already been obtained in a number of
, 1999; Parinov
, 1999; Tissier
, 1999; Ogarkova
The initial stage of gene identiﬁcation in the
insertion mutants was on the whole
related to the necessity of ampliﬁcation and cloning
of the nuclear DNA fragments adhering to inser-
tion. The fully deciphered nucleotide sequence of
the genome presented in computer databases, such
as http/kazusa.or.jp, http://www.arabidopsis.org/,
http://www.ncbi.nlm.nih.gol, makes it possible to
identify the gene, into which an insertion was incor-
porated, according to the sequence of a small, 100
to 200 bp, DNA fragment near the insertion.
The aim of this work is to identify, on the basis of
this strategy, the gene in
, the insertion into
which in the homozygous state leads to a slower devel-
opment of germlings, cessation of their growth at the
stage of cotyledons, and death at the age of two to four
MATERIALS AND METHODS
Line 27 from the collection of
morphological insertion mutants was used,
which belongs to the phenotypic class of recessive
lethal germlings. This collection was previously
obtained using a modiﬁed method of agrobacterial
transformation of germinating seeds (Ogarkova
, 1999) with the help of binary vec-
tor systems pLD3 (Gapeeva
, 1995) and
, 1994). The modiﬁcation com-
prised ultrasound treatment of seeds in the presence of
aluminum oxide prior to cocultivation with agrobacte-
ria, which allowed us increase threefold the efﬁciency
of induction of insertion mutations.
After cocultivation with agrobacteria, the seeds
were germinated aseptically in the soil and all plants
were harvested without selection. Transformed plants
were selected in the next generation (T2) by sterile
seeding and growing for 14 days a sample of mixed har-
vest of T1 seeds on Petri dishes with a synthetic
medium containing the corresponding marker antibiot-
ics. The germlings that were not discolored were then
transferred onto dishes with antibiotic-free medium.
Within 15 to 20 days of growth on the dishes, the plants
were transferred into test tubes under sterile conditions.
Seeds were collected individually from each fertile
plant within 9–10 weeks.
Analysis of the area of A. thaliana genomic DNA
adhering to t-DNA insertion.
A fragment of genomic
DNA adhering to t-DNA insertion was ampliﬁed using
the method of TAIL-PCR (Liu and Whittier, 1995) and
standard kits of reagents (Promega, USA). A system of
three successive primers to the left border DNA area of
Identification of the Gene Whose Mutation Leads
to the Appearance of Recessive Lethal Germlings
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 June 9, 2004; in ﬁnal form, October 26, 2004
—The data are presented on genetic and molecular-genetic analysis of a mutant from the collection
of morphological insertion mutants of
we obtained earlier, which belongs to the pheno-
typic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identiﬁed, which
adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using
: insertion mutant, recessive lethal germling, nucleotide sequence, gene.