ISSN 1062-3604, Russian Journal of Developmental Biology, 2016, Vol. 47, No. 6, pp. 359–366. © Pleiades Publishing, Inc., 2016.
Original Russian Text © T.A. Kritskaya, A.S. Kashin, V.A. Spivak, V.E. Firstov, 2016, published in Ontogenez, 2016, Vol. 47, No. 6, pp. 386–394.
Features of Clonal Micropropagation of Silene cretacea
(Caryophyllaceae) in in vitro Culture
T. A. Kritskaya*, A. S. Kashin**, V. A. Spivak, and V. E. Firstov
Chernyshevsky Saratov National Research State University, ul. Astrakhanskaya 83, Saratov, 410012 Russia
Received March 21, 2016; in final form, May 20, 2016
Abstract—The features of the formation of microshoots in in vitro culture of Silene cretacea—endangered spe-
cies with narrow ecological amplitude, which is a promising source of medicinal raw materials—were studied.
It was demonstrated that, at the micropropagation step, basic Woody Plant Medium containing vitamins
according to Murashige and Skoog and supplemented with 0.2 mg/L 6-benzylaminopurine, 1.0 mg/L kine-
tin, 1.0 mg/L gibberellic acid, and 0.5 mg/L indole-3-acetic acid is the most effective. The combination and
concentration of these growth regulators, selected using mathematical combinatorial analysis, activated axil-
lary buds and provided a high multiplication factor (9.3 ± 1.3 microshoots per explant). Morpho-histological
analysis revealed the main stages of the formation of microshoots and proved the absence of callus formation
during the whole time of the cultivation of explants. The features of the dynamics of the culture during the
year of continuous cultivation are presented.
Keywords: Caryophyllaceae, Silene cretacea, clonal micropropagation, in vitro, rook polynomial, morpho-
Silene cretacea Fisch. ex Spreng., 1825 (Caryophyl-
laceae) is an endemic calcicole dwarf semishrub
(Tsvelev, 2004). It is included in the Red Book of the
Russian Federation (Devyatov, 2008) and in Appendix I
of the Bern Convention (Bilz et al., 2011). In the Sara-
tov oblast, the only population of the species was dis-
covered in 2008 (Nevsky et al., 2009). Prior to this, the
species had not been observed in the oblast for more
than 150 years. Due to low seed production and the
small number of specimens in the detected population,
in vitro clonal micropropagation is the most appropri-
ate method for mass production of planting material of
this species in order to restore its population.
S. cretacea is a promising source of compounds
from the phytoecdysteroids class with a wide spectrum
of physiological activity, low toxicity, and lack of hor-
monal action on mammals (Tuleuov et al., 2014). It
was proven that phytoecdysteroids have antifungal,
anabolic, hypoglycemic, hepatoprotective, and tonic
effects (Báthori et al., 2008).
The only way of mass production of planting mate-
rial for the introduction of this species into culture
with the prospect of pharmaceutical use is the method
of clonal micropropagation.
Recently, many studies were devoted to the devel-
opment of biotechnological approaches for the pro-
duction of plant material of Caryophyllaceae species
(Cheng et al., 2008, etc.); the main goal of these stud-
ies in most cases was the production of callus culture.
However, initial structures preferred for clonal micro-
propagation are meristematic tissues of plants, since
they, unlike the callus, remain genetically stable over
multiple subcultures (Butenko, 1999). This is a key
factor during cultivation of plants in order to preserve
biodiversity or cultivation for breeding or business
Several studies were devoted to in vitro cultivation
of S. cretacea for clonal micropropagation. For micro-
propagation of S. cretacea, E.M. Vetchinkina et al.
(2012) used Murashige and Skoog medium (MS;
Murashige and Skoog, 1962) with twice reduced salt
concentration and the addition of 0.5 mg/L 6-benzyl-
aminopurine (BAP) or 2-izopentyladenine (2-ip).
O.I. Zholobova (2012) demonstrated the possibility of
cultivation of S. cretacea on full MS medium with
0.5 mg/L BAP. However, when S. cretacea was cul-
tured on media suggested by these authors, only indi-
rect organogenesis was observed. The viability of
explants gradually decreased and necrosis processes,
overhydration, and the arrest of morphogenesis pre-
vailed by 3–4 passages. The proportion of dead
explants was 54–56% per one passage, and the maxi-
mum net reproduction was not higher than three. In
the sixth passage, the culture completely dropped out