1022-7954/01/3704- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 4, 2001, pp. 392–400. Translated from Genetika, Vol. 37, No. 4, 2001, pp. 494–503.
Original Russian Text Copyright © 2001 by Tomilova, Tomilov, Ogarkova, Tarasov.
In recent years, the identiﬁcation of
genes has been the focus of intense research. Progress
in this ﬁeld is due to the development of efﬁcient meth-
ods of producing the insertion mutants using either
plasmids integrating into the
genome during genetic transformation or transposons.
As a result, several large collections of
insertion mutants were obtained; further investigations
are in progress. The complete sequencing of
genome and the development of PCR procedures that
allow one to amplify DNA fragments of unknown
sequence adjacent to the insertion dramatically changed
the strategy of gene identiﬁcation. To determine the pri-
mary structure and location of the entire gene by data-
base analysis, it is sufﬁcient to sequence a relatively
small DNA region adjacent to the insertion.
Earlier, we reported the identiﬁcation of
gene involved in regulation of root development . In
this paper, we describe the identiﬁcation of a mutant
gene causing the appearance of necrotic lesions on the
MATERIALS AND METHODS
A mutant selected from T2 seeds
obtained from the transformed T1 seeds of
(Köln, Dijon) grown in the soil was used. A hypervir-
ulent A-281 strain of
rying the pLD3 plasmid derived from the pBI121 plas-
mid, which is a constituent of a binary transformation
vector, was employed .
Media and cultivation conditions.
T2 were germinated under the sterile conditions on a
Kvitko agar medium (0.8%)  without sugars and
vitamins. After surface sterilization , seeds (400 seeds
per Petri dish) in 0.15% agar solution (Serva) were
transferred to the Kvitko medium, containing the
marker antibiotic kanamycin (Km) at a concentration
g/ml. Petri dishes were kept in the dark for two
days at 4
C and then transferred to a 16-hour light day
Selection of transformants in T2.
To select kanamy-
cin-resistant plants, T2 seedlings were cultured on anti-
biotic-containing medium for 14 days. Seedlings that
remained green were transferred to a Km-free medium.
After growth on this medium from 15 to 20 days, each
plant was placed in a separate tube and cultured from
nine to ten weeks, whereupon mature seeds were col-
lected from the fertile plants (T3 generation).
Analysis of segregation for plant phenotype.
phenotypically mutant forms, segregation analysis was
conducted in T3, T4, and T5 progenies in a series of
consecutive experiments. Seeds (10–50 seeds from one
pod) were sterilized for 15–20 min in 6% hydrogen per-
oxide, washed twice with sterile water, and transferred
in 0.15% agar solution to a Km-free medium. After
5–8 days, seedlings were analyzed for the mutant phe-
notype manifested in necrotic cotyledons and changed
growth rate. Two-week-old plants were transferred to
the tubes with growth medium or planted in the soil.
The embryonic analysis was
carried out by standard procedures .
Histochemical staining for
The GUS activity in plant tissues was mea-
sured according to Jefferson
 using X-GlcA
(Sigma). Tissues (20–50 mg) were incubated in 1 mM
X-GlcA solution in 50 mM phosphate buffer, pH 7.0,
for 12–36 h at 37
C. The development of blue staining
indicated expression of the marker
Identification of a Mutant Gene Controlling Necrotic Cotyledons
in Developing Seedlings of
N. B. Tomilova, A. A. Tomilov, 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 October 13, 2000
—Genetic and molecular analyses of an
mutant with necrotic cotyledons from
the collection of insertion mutants obtained earlier were conducted. The mutation under study showed incom-
plete dominance and represented a single insertion of the T region of pLD3 vector used for transformation of
germinating seeds to the plant genome during the creation of the collection. Using TAIL–PCR, a fragment of
the mutant DNA adjacent to the left border of the T-DNA insertion was isolated and sequenced. Computer
r-aided analysis showed that the insertion was located on the left arm of chromosome 1. The open reading frame
containing the insertion has one exon and encodes a protein of 446 amino acids, whose function is unknown.