1022-7954/01/3701- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 1, 2001, pp. 30–38. Translated from Genetika, Vol. 37, No. 1, 2001, pp. 36–45.
Original Russian Text Copyright © 2001 by Tomilov, Tomilova, Ogarkova, Tarasov.
Insertion mutagenesis is currently used for studying
functional organization of both pro- and eukaryotic
genes. Pro- and eukaryotic transposable elements are
used as insertions for isolation and subsequent analysis
of genes [1–3]. In the case of
T-DNA insertions transferred into the plant genome by
means of agrobacterial transformation are widely used.
This method is suitable because
has a small
genome and a low proportion of repetitive DNA
sequences [4, 5]. Since almost entire
genome has been sequenced, the insertion mutagenesis
allows to identify genes without cloning and subse-
quent complete sequencing. It is sufﬁcient to determine
the nucleotide sequences of relatively short DNA frag-
ments near the insertion sites and identify these frag-
ments using computer databases [6–8].
We earlier generated a collection of
insertion mutants by means of agrobacterial transfor-
mation. Based on this collection, we selected a lethal
root mutant characterized by a shortened hypocotyl. In
the present study, we present data on isolation and char-
acterization of this mutant as well as on identiﬁcation
of the corresponding gene.
MATERIALS AND METHODS
We used a lethal root mutant of
ecological race Köln. This mutant was
selected from seeds of generation T2 obtained from
transformed T1 seeds germinated and grown in soil.
 carrying the plasmid pLD3 was used for transfor-
mation. The plasmid pLD3 is a derivative of the plas-
mid pBI121, a component of the binary vector system
of plant transformation. Its T-region contains, along
with the marker neomycin phosphotransferase (NPTII)
-glucuronidase (GUS) genes (under the promoter
of the nopalin synthase gene and promoter CaMV35S,
respectively), the gene determining the resistance to the
antibiotic chloramphenicol expressed in
Media and cultivation conditions.
Seeds of T2 gen-
eration were germinated under sterile conditions in
Kvitko’s medium  containing 0.8% agar. The
medium was vitamin- and sucrose-free and contained
the marker antibiotic kanamycin (Km). Four hundred
seeds were planted on each dish. After planting, the
dishes were left in the dark at 4
C for two days and then
cultivated at a photoperiod of 16 h and temperature
Isolation and analysis of DNA.
DNA was isolated
from plant tissues and puriﬁed according to Rogers and
Bendich . We used standard molecular-cloning
methods . Promega assay kits were used for PCR
analysis of genomic DNA  and sequencing of DNA
Selection of transformants in generation T2.
plants for the resistance to the marker
antibiotic Km (50
g/ml), T2 seedlings were incubated
on the Km-containing medium for 14 days. After this,
the seedlings that remained colored were transferred to
a Km-free medium. After 15–20 days of growth on this
medium, each plant was transferred into a separate test
Identification of a Gene Involved
in the Control of the Root System Development
A. A. Tomilov, N. B. Tomilova, O. A. Ogarkova, and V. A. Tarasov
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 117809 Russia;
fax: (095) 135-43-27; e-mail: email@example.com
Received May 23, 2000
—Genetic and molecular genetic analysis of a lethal root mutant of
out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation
was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation
of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment ﬂanking the
left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was deter-
mined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the
gene in chromosome 1.