1022-7954/01/3708- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 8, 2001, pp. 899–904. Translated from Genetika, Vol. 37, No. 8, 2001, pp. 1081–1087.
Original Russian Text Copyright © 2001 by Ogarkova, Tomilova, Tomilov, Tarasov.
Determination of the complete nucleotide sequence
genome performed within
coordinated international research projects was ﬁn-
ished in 2000 [1–4]. This promises a rapid progress in
understanding the functions of genes of this higher-
plant species. Its relatively small genome and a low per-
centage of DNA repetitive sequences permits to apply
in this species insertion mutagenesis  using both
direct and reverse genetic approaches .
Development of a representative collection of inser-
tion mutants is a necessary condition for application of
direct and reverse genetics. Therefore, the aim of the
present study was to obtain and describe a collection of
morphological mutants bearing T-DNA
MATERIALS AND METHODS
We used two
races: Köln and Dijon.
Transformation of germinating seeds was performed
by the Feldmann method modiﬁed as in [7, 8] with the
. The vectors pLD3  and pPCVRN4  were
Activity of the reporter
gene was assayed according to the Jefferson histochem-
ical method modiﬁed as in  with X-GlcA dye pur-
chased from Sigma.
Polymerase chain reaction conditions for rapid
determination of T-DNA in transformed plants and
primers for the T-DNA pLD3 vector were described in
our previous study . The primers Cb-1: (5'-
GGTTTCTTAGACGTCAGGTG-3') and Cb-2: (5'-
CGCCTCCATCCAGTCTATTA-3') were used in
experiments with the vector pPCVRN4. They allowed
ampliﬁcation of a 778 kb long DNA fragment of the
gene in the T-DNA.
Phenotype segregation of the progeny was analyzed
in successive experiments in the T3, T4, and T5 gener-
The selective antibiotics used were kanamycin
(50 mg/l) and hygromycin (30 mg/l).
A medium with 0.5 mg/l 6-benzylaminopurine and
-indolyl-3-butyric acid was used for saving
lethal mutants and compensation of possible reproduc-
tion disorders in sterile mutants.
RESULTS AND DISCUSSION
Transformation of Plants
Insertion mutations were induced by the modiﬁed
-mediated genetic transfor-
mation of germinating seeds ﬁrst proposed by Feld-
mann and Marks . A modiﬁcation involving ultra-
sound sonication of seeds with the presence of alumina
prior to cocultivation with
resulted in a
more than threefold increase in the efﬁciency of inser-
tion mutagenesis [7, 8].
Transformed plants were selected in the progeny
following transformation (T2), when a random sample
of T1 seeds was germinated on a synthetic medium
containing an appropriate marker antibiotic (Fig. 1).
Transformation was performed with two binary vec-
1. The vector pLD3 (Fig. 2) is a derivative of the
vector pBI121. Its T-DNA contains the
neomycinphosphotransferase II imparting kanamycin
Morphological Insertion Mutants
O. A. Ogarkova, N. B. Tomilova, A. A. Tomilov, and V. A. Tarasov
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991, Russia;
fax: (095)1354327; e-mail: email@example.com
Received January 30, 2001
—A collection of transgenic
plants has been obtained by
ated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids
pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of
genes. Seventy-ﬁve lines have been chosen from the collection and subjected to genetic and molec-
ular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various mor-
phological defects. Identiﬁcation of mutant phenotypes and genetic characterization of the transgenic lines have
been performed with the use of nutrient media supplemented with exogenous hormones, which revealed ﬁve
recessive lethal mutants and one dominant sterile mutant.