1022-7954/05/4109- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 9, 2005, pp. 985–990. Translated from Genetika, Vol. 41, No. 9, 2005, pp. 1203–1209.
Original Russian Text Copyright © 2005 by Pukhnacheva, Novoselya, Zotkevich, Deineko.
The method of agrobacterial transformation has
been widely employed in recent years for creating
transgenic plants . As a vector upon agrobacterial
transfer, T-DNA, i.e., a region of Ti-plasmid from the
two imperfect direct repeats of 25 bp, was used. The idea
that only T-DNA situated between border repeats is
transferred into the plant cell was widely accepted over a
long period of time [2, 3]. However, proceeding from the
results reported in recent years, it is obvious that frag-
ments of the “outer” (vector) DNA can be also trans-
ferred and stably integrated into the plant genome [4–7].
The reasons for such transfer may be related to T-DNA
processing errors in the agrobacterial cell [2, 8, 9].
T-DNA is processed by gene products in the Vir
region of Ti-plasmid. The processing is initiated when
the protein VirD2 (endonuclease) produces (by the aid
of VirD1) a single-stranded nick in the region of termi-
nal T-DNA repeats and brings about covalent binding
with 5' end of T-DNA. Replication of the new T-DNA
strand occurs in the direction from the right terminal
repeat toward the left, and this strand gradually replaces
the old one. The released T-DNA strand, as a single-
stranded molecule bound along the whole molecule
with proteins VirE2 and with VirD2 at 5' end, is trans-
ferred into the nucleus of plant cell and becomes inte-
grated into the plant nuclear genome [2, 3].
It has been ascertained that errors occurring at all
described stages of T-DNA processing may cause exci-
sion of not only T-DNA but also vector DNA portions
[4–7]. In a number of cases, imprecise excision of a T-
DNA fragment was observed, namely, when the frag-
ment is excised from the plasmid lacking the VirD2
protein  or when the protein may have difﬁculty rec-
ognizing boundaries of the T-DNA region .
Sequences of inner T-DNA regions adjacent to terminal
repeats also play an important role in the processing.
Elimination of these regions and their subsequent sub-
stitution with DNA fragments carrying a random
sequence of nucleotides signiﬁcantly increased the
number of errors occurring upon recognition of T-DNA
Fragments resulting from errors during T-DNA pro-
cessing differ in the extent and contain both the entire
vector sequence and certain parts of this sequence
[5, 10, 11]. These fragments are integrated into the
plant genome together with T-DNA or irrespective of
this DNA [4, 7, 12].
Prokaryotic sequences are recognized in the plant
genome as foreign and become methylated , thus
affecting the expression stability of transferred genes
[14, 15]. It has been ascertained that reiterated regions,
or the replication origin (
) sites contained in plasmid
DNA may stimulate rearrangements within the T-DNA
insertion [16, 17]. The transfer into the plant genome of
genes for antibiotic resistance and of agrobacterial vir-
ulence genes, components of the vector sequence
(when using nonbinary vectors), also seems to be unde-
Thus, with consideration of the available data, we
may conclude that undesirable fragments of the vector
DNA can be transferred into the plant genome as a
result of incorrect T-DNA processing during agrobacte-
rial transformation. In accordance with the goal of this
work, the veriﬁcation of the rate with which transgenic
plants carrying in the genome the vector DNA frag-
ments can be detected is currently an urgent problem
and should be further pursued using two specimens of
randomly isolated transgenic tobacco and carrot plants.
MATERIALS AND METHODS
tobacco plants containing marker
genes for neomycin phosphotransferase (
) and carrot plants containing
together with the target gene of
human interleukine-18 [18, 19] obtained by a standard
Insertion of Vector Sequences
in the Genome of Transgenic Plants
N. V. Pukhnacheva, T. V. Novoselya, E. A. Zotkevich, and E. V. Deineko
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia;
fax: (3832)33-12-78; e-mail: firstname.lastname@example.org
Received October 25, 2004; in ﬁnal form, February 25, 2005
—Insertion of vector sequences of different lengths in the genome of transgenic tobacco and carrot
plants occurring at a frequency of 35.2% was shown. No signiﬁcant differences in insertion frequencies
between the two species were observed. Integrated fragments of the vector DNA were stably inherited in the
following generation resulted from self-pollination of original transformants.