1021-4437/03/5003- $25.00 © 2003
Russian Journal of Plant Physiology, Vol. 50, No. 3, 2003, pp. 369–373. Translated from Fiziologiya Rastenii, Vol. 50, No. 3, 2003, pp. 415–419.
Original Russian Text Copyright © 2003 by Karanova, Redichkina, Butenko.
To proliferate and grow
, plant cells need
phytohormones supplemented to a nutrient medium.
However, there are strains and clones that grow without
exogenous auxins or cytokinins [1–7]. Biologists have
been long ago intrigued by the nature of the habituation
trait, in particular, auxin independence. Spontaneously
strains maintained the
habituated state while cells grew in
lost this trait following plant regeneration and recloning
; this evidence points out at the epigenetic origin of
this trait. This aspect has not been studied in the strains
Benth. selected on the hormone-free medium following
mutagen treatment [3, 5].
In the present study we obtained a tobacco cell line
with a heritable habituation trait and studied cell
growth in this line.
MATERIALS AND METHODS
Young callus culture with a potential for morpho-
genesis obtained by Dr. E.I. Komizerko (Timiryazev
Institute of Plant Physiology, RAS) from parenchyma
pith cells of tobacco (
L., cv. Wis-
consin 38) served an initial material for this study.
From this culture, we derived a rapidly growing cell
suspension, which was further used for cloning and
mutagen treatment. The calli growing on an agar-solid-
iﬁed nutrient medium were subcultured every 4–5-
weeks, and the cell suspension, every the two weeks
In most cases, cells were grown on MS salts  sup-
plemented with 3% sucrose, 80 mg/l meso-inositol,
0.4 mg/l thiamine, 1.0 mg/l IAA, and 1 mg/l kinetin.
This standard medium is abbreviated as A
media free from auxin (A
) and from auxin and kine-
) were used for cell selection.
Cloning was carried out using the method of Berg-
mann . Cell suspension was mixed with an equal
volume of the nutrient medium containing 1.6% agar at
C to the initial density of 5
cell/ml and trans-
ferred into petri dishes. The reproductive viability, or
plating efﬁciency, was deﬁned as the percentage of via-
ble colonies of the total number of plated living cells.
1 mm in diameter were scored, because pre-
viously we found that only such colonies proliferate
further under similar conditions . Following four
weeks of culturing, separate colonies were subcultured
onto fresh standard or selective media.
This cell suspension was treated with a chemical
mutagen N-nitroso-N-methylurea (N-NMU) at the con-
centration of 4 and 8 mM for 1 h, rinsed ﬁve times with
the excess of fresh nutrient medium, and plated into
petri dishes. The standard medium was used to score
viability and the selective medium, to score and select
habituated cells. The mutagen was synthesized at the
Institute of Chemical Physics, RAS (Moscow).
The initial strain and the selected auxin-independent
cell line were grown on the agar-solidiﬁed and in liquid
nutrient media. In the latter case, cells were cultivated
in 0.5-l ﬂat-bottomed ﬂasks, with inoculum (1 g fr wt)
added to 60 ml of liquid medium. Cells were subcul-
Selection of a Tobacco Auxin-Habituated Cell Line
and Its Growth Characteristics
S. L. Karanova, T. D. Redichkina, and R. G. Butenko
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;
fax: 7 (095) 977-8018; e-mail: email@example.com
Received September 27, 2001
—Culturing conditions and the dose of a chemical mutagen N-nitroso-N-methylurea were worked out
for selecting auxin-independent (auxin-habituated) cell lines of tobacco (
L.). A clone (strain
B-4) that steadily grew on an auxin-free nutrient medium and did not express any traits of morphogenesis
emerged at the frequency of 1 per 7
plated cells. Mutation was the cause of habituation in this cell line as
corroborated by a low frequency of its emergence, the appearance of the habituation trait only after the
mutagenic treatment, and the stability of habituation trait for many years when the cells were subcultured in
both selective and nonselective media. The auxin-habituated strain B-4 grew more rapidly on an auxin-free
medium, was cytokinin-dependent, resistant to high exogenous IAA concentrations, and incapable of morpho-
Key words: Nicotiana tabacum - N-nitroso-N-methylurea - selective media - habituation
: MS—Murashige and Skoog nutrient medium;