1022-7954/01/3706- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 6, 2001, pp. 643–648. Translated from Genetika, Vol. 37, No. 6, 2001, pp. 784–790.
Original Russian Text Copyright © 2001 by Zagorskaya, Deineko, Sidorchuck, Shumnyi.
It is known that exogenous DNA (T-DNA) trans-
ferred by agrobacteria may be inserted in transcription-
ally active regions of the plant genome and alter plant
gene functioning . This possibility termed T-DNA-
induced mutagenesis  was experimentally conﬁrmed
. Numerous T-DNA-induced muta-
tions are described: alterations of plant height [2, 4],
size and shape of leaf blades  and ﬂower and inﬂo-
rescence structure [6, 7], embryonic lethality, and male
sterility [8, 9]. We have previously described mutations
that altered ﬂower morphology and caused male steril-
ity in transgenic tobacco plants .
The data available in the literature on T-DNA-
induced mutations indicate that these mutations can be
classiﬁed into two groups depending on their inherit-
ance. Mutations coinherited with the marker genes
incorporated into T-DNA insertions (for instance, kan-
amycin-resistance gene) constitute the ﬁrst group.
These mutations result from T-DNA insertion in the
regions of genes controlling the expression of the
altered phenotypic traits. The marker-gene-nonlinked
T-DNA-induced mutations contribute to the second
group. The origin of these mutations still remains
unknown, and therefore, it was interesting to study the
mode of inheritance (linked or nonlinked) of some
traits, such as resistance to antibiotic kanamycin and
alterations of ﬂower morphology, in previously
obtained transgenic tobacco plants.
MATERIALS AND METHODS
In this study, we used 15 transgenic plants T
have altered ﬂower morphology [10, 11] and were pre-
viously obtained in various experimental series with
the use of different genetic constructs (Table 1). Trans-
genic nature of the parental plants was conﬁrmed by
ampliﬁcation of the DNA fragments corresponding to
the transferred DNA in polymerase chain reaction
using plant DNA as a template.
The changes in ﬂower morphology in the studied
plants manifested in longostylia (elongated pistil and
shortened ﬁlaments) or the formation of wavy corolla
and expanded pistil stigma. In individual plants, either
all these traits or their combinations were present.
The mutant phenotype was visually determined in
plants of the ﬁrst and second generations (T
respectively) obtained by compulsory self-pollination
of the original transformants upon preliminary ﬂower
isolation. After self-pollination, the progeny was tested
for resistance to the antibiotic using selective medium
. Kanamycin-resistant plants were transferred into
a hydroponics greenhouse and grown at 20/15
(day/light) temperature regimen and photoperiod of
16/8 h. Pollen fertility was assessed by a conventional
method, namely, microspore staining with acetocar-
mine . As previously accepted , plants with pol-
len fertility higher than 50% (control level correspond-
ing to that of the SR1 nontransgenic line) were consid-
ered fertile. Transformants with male fertility lower
than 50% were considered semifertile. The observed
and expected segregations in T
were compared using the
The level of endogenous hormones and their effect
on the mutant trait expression in the transgenic plants
were evaluated using reciprocal grafting. For this pur-
pose, young shoots were taken prior to generative organ
formation either from nontransgenic plants of the con-
trol line SR1 or from transgenic tobacco plants display-
ing mutant phenotype.
After agrobacterial transfer, one to several indepen-
dent T-DNA insertions occur in the plant genome .
Hence, if even one of them is introduced into a func-
tional region of the genome, a mutant phenotype may
be generated. Cosegregation of the marker gene (resis-
tance to antibiotics) and the mutant traits indicates the
insertional nature of the mutation.
Inheritance of Altered Flower Morphology
and Kanamycin-Resistance in Transgenic Tobacco Plants
A. A. Zagorskaya, E. V. Deineko, Yu. V. Sidorchuck, and V. K. Shumnyi
Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090 Russia; e-mail: firstname.lastname@example.org
Received September 22, 20000; in ﬁnal form, January 25, 2001
—Inheritance of altered ﬂower morphology and resistance to antibiotic kanamycin was studied in the
ﬁrst and second generations (T
, respectively) of self-pollinated transgenic tobacco plants. In most trans-
formants, kanamycin resistance was closely linked to mutant phenotype. T-DNA-induced mutations were
shown to be dominant.