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.
MORPHOGENESIS
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 *e-mail:
[email protected] **e-mail:
[email protected] 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 species 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 kinetin, 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 axillary 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, morphohistological analysis DOI: 10.1134/S1062360416060023
INTRODUCTION Silene cretacea Fisch. ex Spreng., 1825 (Caryophyllaceae) 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 Saratov oblast, the only population of the species was discovered 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 appropriate 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 hormonal 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 material 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 development of biotechnological approaches for the pro-
duction of plant material of Caryophyllaceae species (Cheng et al., 2008, etc.); the main goal of these studies in most cases was the production of callus culture. However, initial structures preferred for clonal micropropagation 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 purposes. Several studies were devoted to in vitro cultivation of S. cretacea for clonal micropropagation. For micropropagation 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-benzylaminopurine (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 cultured on media suggested by these authors, only indirect organogenesis was observed. The viability of explants gradually decreased and necrosis processes, overhydration, and the arrest of morphogenesis prevailed by 3–4 passages. The proportion of dead explants was 54–56% per one passage, and the maximum net reproduction was not higher than three. In the sixth passage, the culture completely dropped out
359
360
KRITSKAYA et al.
(Kritskaya and Kashin, 2013). Empirical selection of MS medium with different variants of phytohormones and their doubles or triple combinations did not give the desired result (Kritskaya et al., 2015). We have demonstrated that the use of Woody Plant Medium (WPM; McCown and Lloyd, 1981) increases the morphogenetic potential of S. cretacea culture in comparison with MS and other variants of media (up to seven microshoots per explant), but the formation of shoots occurs by indirect organogenesis. The optimal concentration of sucrose was identified (20 g/L) (Kritskaya and Kashin, 2013). Later, WPM medium with this concentration of sucrose was used. The goal of this study was the improvement of the efficiency of clonal micropropagation of S. cretacea by direct organogenesis due to the selection of the composition of the culture medium using mathematical modeling methods, the identification of morphogenetic features of formation of microshoots, and the investigation of the dynamics of culture during long subcultivation. MATERIALS AND METHODS Investigations were carried out in 2012–2015 using conventional techniques for cultures of plant tissues and organs (Butenko, 1999). Media containing macro- and micro- salts according to WPM (McCown and Lloyd, 1981), sucrose (20 g/L), vitamins and amino acids as in MS, agar–agar (Panreac, UK) 9 g/L, pH 5.9–6.1 was used as a basic medium (BM). Autoclaving time was 20 min at 121°C. Segments of shoots 1.0–1.5 cm height with 2–3 nodes obtained by cultivation of mature S. cretacea seeds on BM without phytohormones were explanted. Seeds were collected from a natural population growing in the Saratov oblast. Seeds were sterilized sequentially using Pril (Henkel-ERA, Russia) for 30 min, 70% ethanol for 3–5 min, 1.25% NaOCl solution (Electra, Russia). Seeds were then washed five times with sterile distilled water in the laminar flow hood (Lamsystems, Russia) and placed on the growth medium.
formed using the randomization of the configurations of rook dispositions. The medium (20–40 mL) was dispensed into chemical vessels of 100 mL. Five–six explants per vessel were cultured at 24 ± 1°C, 16 photoperiod, and light 2–3 thousand lux. Results were analyzed on the 21th day of cultivation. Hormone-free BM was used as control. The multiplication factor, the shoot length, and weight of callus-like tissues were estimated as main parameters of the culture. The multiplication factor means the amount of microshoots regenerated per one passage per one explant. Morpho-Histological Analysis The material was collected every three days by fixing ten explants in a mixture of formalin, glacial acetic acid, and 50% ethanol in a volume ratio of 4 : 1 : 10 (FAE) for 3 hours. The fixed material was transferred to a glycerol-alcohol solution in the ratio of 1 : 1. Prepared explants were examined under a Stemi 2000-CS (Carl Zeiss, Germany) stereomicroscope. Anatomical sections were prepared using a rotary microtome according to conventional methods (Aescht et al. 2010a). As controls, samples of intact campion plants were used. Ready samples were first stained with hematoxylin according to Heidenhein (Aescht et al., 2010b), then with combined staining according to D. Tolivia and J. Tolivia (Tolivia, D. and Tolivia, J., 1987). Preparations were studied under the microscope Axio Lab A1 (Carl Zeiss, Germany) and photographed using a camera (Canon Power Shot G11, Japan) with an adapter. Statistical Data Processing Experiments were performed in triplicates, and at least 30 explants were selected for each replicate. Statistical data processing was carried out by standard methods using Statistica for Windows, v. 6, and Microsoft Excel software. RESULTS Selection of Growth Regulators
Selection of Growth Regulators BAP, kinetin (KN), gibberellic acid (GA), and indole-3-acetic acid (IAA) at concentrations of 0.2, 0.5, and 1.0 mg/L were used at the stage of selection of phytohormones. Phytohormones were added to the culture medium prior to autoclaving. Mathematical rook polynomial method was used to achieve the desired results (Riordan, 2002). Four-factor, threelevel table, including four phytohormone (BAP, SC, GA, and IAA) with three levels of concentrations of 0.2, 0.5, and 1.0 mg/L was comprised (Table 1), within which the search for the optimal combinations of phytohormones for regeneration of S. cretacea was per-
Maximal values of multiplication factor (9.3 ± 1.3 microshoots per explant) and shoot length (17.5 ± 0.7 mm) were observed for the combination BAP 0.2 + KN 1.0 + GA 1.0 + IAA 0.5 mg/L (Table 1). Change of phytohormones concentration lead to a decrease in activity and proliferation of organogenic callus-like tissues. A strong positive correlation between the multiplication factor and the shoot length (r = 0.83; р ≤ 0.05) and a negative correlation between the multiplication factor and the shoot length, on the one hand, and the weight of callus-like tissues, on the other hand, (r = –0.78 and r = –0.81, respectively; р ≤ 0.05) (Table 2) were revealed.
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 47
No. 6
2016
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 47
No. 6
2016
KN 0.5
IAA 0.2
K 4.5 ± 0.5 L 11.7 ± 0.6 M 1.0 ± 0.6
K 3.0 ± 0.3 L 9.1 ± 0.6 M 1.1 ± 0.1
KN 0.2
K 3.8 ± 0.6 L 9.0 ± 0.6 M 1.2 ± 0.2
K 4.7 ± 0.4 L 12.8 ± 1.2 M 0.9 ± 0.1
KN 1.0
IAA 0.5
K 5.1 ± 1.0 L 14.3 ± 1.2 M 0.7 ± 0.1
K 5.1 ± 0.8 L 15.5 ± 1.1 M 0.9 ± 0.1
K 5.7 ± 0.5 L 13.4 ± 0.7 M 0.8 ± 0.1
KN 0.5
K 3.3 ± 0.5 L 9.5 ± 0.8 M 1.1 ± 0.2
K 5.6 ± 0.6 L 14.7 ± 0.8 M 0.7 ± 0.1
K 5.4 ± 0.5 L 12.3 ± 0.9 M 0.9 ± 0.1
KN 0.2
K 3.3 ± 0.3 L 9.9 ± 0.7 M 1.0 ± 0.1
K 5.4 ± 0.6 L 15.7 ± 0.9 M 0.7 ± 0.1
K 5.7 ± 0.6 L 13.6 ± 0.8 M 0.6 ± 0.1
K 9.3 ± 1.3 L 17.5 ± 0.7 M 0.6 ± 0.1
KN 1.0
BAP 1.0
IAA 1.0
IAA 0.2
IAA 0.5
IAA 1.0
IAA 0.2
IAA 0.5
K 5.4 ± 1.0 L 15.9 ± 1.2 M 0.7 ± 0.1
KN 0.5
BAP 0.5
K 2.9 ± 0.3 L 8.8 ± 0.6 M 1.3 ± 0.1
K 3.7 ± 0.7 L 10.3 ± 0.7 M 1.2 ± 0.2
KN 1.0
p ≤ 0.05 for confidence intervals; K, multiplication factor, the number of microshoots per explant; L, shoot length, mm; M, the weight of callus-like tissues, g. Values were recorded on 21 days of the cultivation.
GA 0.2
GA 0.5
GA 1.0
IAA 1.0
KN 0.2
BAP 0.2
Table 1. Morphogenetic response of S. cretacea explants to various combinations of phytohormones and their concentration (mg/L). The table is presented in the form of a chessboard in accordance with the theory of rook polynomials (Riordan, 2002). Semibold font indicates first configuration of rook disposition; regular font indicates reiterated dispositions
FEATURES OF CLONAL MICROPROPAGATION 361
362
KRITSKAYA et al.
Table 2. Results of the correlation analysis (Pearson’s coefficient r) between morphometric parameters of the culture and the concentration of phytohormones Correlation with concentration
Multiplication factor Shoot length Weight of the callus type tissue
BAP
KN
GA
IAA
–0.75853 –0.88199 0.894427
0.280939 0.019938 0.255551
0.674253 0.88785 –0.55902
0.030101 0.050433 –0.3993
р ≤ 0.05. Each phytohormone was taken in concentrations 0.2, 0.5, and 1.0 mg/L.
Since the main objective of our study was to obtain high-quality and high-grade regenerated S. cretacea, a combination BAP 0.2 + KN 1.0 + GA 1.0 + IAA 0.5 mg/L, which we arbitrarily designated as “SCS,” was chosen as optimal variant (Kritskaya et al., 2015). Dynamics of the Culture During the year of the continuous cultivation on the SCS medium, a decrease of the multiplication factor was observed twice: the first negative peak was in July, and the second covered the period from October to January (Fig. 1). Despite the constant cultivation conditions (SCS medium, temperature +24 ± 1°C, photoperiod 16/8) in the indicated periods, a decrease in the multiplication factor from 11.9 ± 1.3 (in the period of active growth) to 7.3 ± 0.7 microshoots per the explant was detected. Full growth arrest processes, i.e., lack of growth in the length of the shoots, was observed when the S. cretacea culture was kept at low positive temperatures (+5 ± 1°С) on the hormone free
BM medium from October up to and including January. After 4 months at a lower temperature and the return of explants in standard culture conditions, growth resumed in 2 weeks and the multiplication factor increased up to 15.0 ± 1.4 microshoots per explant. In case of further cultivation of explants under low positive temperature, dormancy could be extended up to 6 months without damage to the plants. Longer exposure, for example, during the year, led to the deaths of approximately 50% of explants, and the remaining 50% lost the ability to morphogenesis. The transfer of explants in the phase of active growth under conditions of low temperature (e.g., from August to October) caused slowing of growth processes, but they did not stop completely. Growth during this period was approximately 1/3 of the initial shoot length. The growth stopped by mid-October. Morphogenesis During the first week of cultivation on SCS medium, activation of growth process in the nodal
Multiplication factor (number of shoots/explant) 16 14 12 10 8 6 4 2 0 May
July June
Sept. Aug.
Nov. Oct.
Jan. Dec.
Mar. Feb.
Apr.
Fig. 1. Dynamics of S. cretacea culture on SCS medium during the year of continuous subcultivation. RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 47
No. 6
2016
FEATURES OF CLONAL MICROPROPAGATION
(a)
150 µm
363
500 µm
(b)
200 µm
(c)
250 µm
(d)
5 mm
(f)
(e)
1 mm
(g)
5 mm
Fig. 2. Stages of morphogenesis of S. cretacea on SCS medium: (a) the development of axillary buds in the nodal region (tangential section of nodal region on the 7th day of cultivation); (b) the growth of axillary buds in the second node of the explant on the tenth day of cultivation (tangential section of the peripheral part of the explant stem); (c, d) regenerated shoots with (c) normal structure and (d) fasciation on the 16th day of cultivation (objects were photographed in glycerol-ethanol solution); (e) the rapture of nodal region of the explant and passage of parenchymal cells from the place of rapture (longitudinal section of the stem part of the explant on the 16th day of cultivation); (f, g) the formation of buds on the edge of the rupture (longitudinal section).
region of explants and the development of one or two buds in the base of the leaves was observed (Fig. 2a). On the 10th day, active growth of lateral shoots of the second order and the formation of 3–4 buds in the bottom of the shoots between the leaf and stem was detected. An increase in the size of buds located in the axils of primordia in the first and the second nodes was noted (Fig. 2b). On the 13th day, the growth of shoots of the third order and growth of the nodal region was observed. On the 16th day, shoots formed by this time could be divided into two groups: one formed the stem RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
with the typical structure (82.8 ± 8.1%) (Fig. 2c) and another (17.2 ± 1.3%) with fascial shape of the stem, which remained in shoots of the first order, while microshoots of following orders extended from it had normal structure (Fig. 2d). At this point, in the nodal region of the explant, active proliferation of cells of main parenchyma occurred, which lead to the rupture of the epidermis (Fig. 2e). By the end of the third week of cultivation, with increasing size of microshoots, proliferation of parenchyma cells in the rupture of the nodal region of the explant was accompanied by the Vol. 47
No. 6
2016
364
KRITSKAYA et al.
proliferation of peripheral groups of cells. On the edges of the rapture, the formation of many bud-like structures with differing degrees of development was observed (Figs. 2f, 2g). Bud-like structures formed in such a way continued to grow after transfer to fresh medium without phytohormones. The number of microshoots received during the second passage was 5.6 ± 0.4 pcs per secondary explant. Thus, a sufficient amount of microshoots ready for the next stage of rooting formed after 3 weeks of cultivation. DISCUSSION Studies have shown that the medium with mineral WPM basis and organic additives according to MS, supplemented with BAP 0.2 + KN 1.0 + GA 1.0 + IAA 0.5 mg/L (SCS), is optimal for micropropagation of S. cretacea. It does not reduce the morphogenetic potential of plant tissues under continuous cultivation. Application of the methods of combinatorial analysis, in particular the rook polynomial method (Riordan, 2002), allowed us to reduce the number of experimental options from 81 to 9 and choose the best option. Values were scattered randomly on the “chessboard” and formed a “Gaussian spot” with an area of the optimum, including BAP 0.2 + KN 1.0 + GA 1.0 + IAA 0.5 mg/L point, around which the less efficient similar values and pessimal interval with variants supplemented with BAP 1.0 mg/L and GA 0.2 mg/L were grouped. Permutations of rook polynomial configuration confirmed our data (Table 1). A similar approach (Latin square method) was used previously to optimize the mineral composition of the growth medium for in vitro cultivation of wheat apexes (Spivak, 1994). The obtained results can be fully explained by the idea about the action of different groups of phytohormones on the plant. It is known that morphogenetic response to exogenous growth regulators is due, primarily, to the genotype of the object; thus, even different populations of one species may respond differently to the same conditions (Mitrofanova, 2011). Cytokinins (BAP, KN) have multifunctional effects on physiological processes in higher plants. The most typical cytokinin effects on in vitro culture are the suppression of apical dominance and stimulation of lateral buds, mainly due to the differentiation of vascular tissue between the axillary buds and vascular bundles of the main stem (Van Staden et al., 2008). It was noted that the introduction of several cytokinins simultaneously in the growth medium improves the quality and quantity of regenerated shoots for some crops (Yu et al., 2012). The main property of GA is the stimulation of cell division and elongation. However, primarily cell division is activated under the influence of GA. Due to this, there is an increase in net reproduction and the shoot length. IAA in combination with GA enhances the action of the latter (George et al., 2008). Kinetin, together with auxin (IAA), participates in the organogenesis process in plants. In the classic work of Skoog
and Miller (Skoog and Miller, 1957) on a culture of undifferentiated tissue of tobacco stem callus, it was found that the presence of 2 mg/L IAA and 0.02 mg/L KN is required for formation of roots. Increasing of KN concentration to 0.5–1.0 mg/L, on the contrary, lead to the induction of the formation of pedunculate buds. IAA alone (0.5–2.0 mg/L) caused tissue expansion, and KN had no apparent effect on the process of differentiation of tissue and organs. In our study, the most effective concentrations of KN and IAA were 1.0 and 0.5 mg/L, respectively. However, as the correlation analysis shown, BAP and GA played the main role in the formation of microshoots of S. cretacea. High concentrations of BAP (0.5–1.0 mg/L) caused impairments to development, the growth of callus, and overhydration of explants, whereas low concentrations of GA (0.2–0.5 mg/L) were insufficient for the regulation of division and expansion of tissues. Negative peaks of multiplication factor values observed in the experiment, by dates, correspond to phenological phases of physiological (summer and winter) dormancy of donor S. cretacea plants associated with the experience of adverse periods (Polevoi and Salamatova, 1991). It is known that the full deep dormancy period in most cases is short. Immediately after this, an induced dormancy period follows, which is caused only by low temperatures (Libbert, 1993). Earlier, a decrease of net reproduction was detected for in vitro culture of wild orchids of temperate latitudes (Ponert et al., 2011) during clonal micropropagation in mid-summer and autumn; it occurred regardless of external factors upon the approach of the respective phenological phases. Thus, in accordance with the above, the total arrest of growth of explants in vitro occurred under the effect of endogenous (dormancy of meristems) and exogenous (low temperature) factors together. Individually, these factors contributed to slower growth processes of in vitro culture of S. cretacea but not to the complete arrest. Based on the results, during phenological phases of deep dormancy (from October to January), it is recommended to maintain a culture of S. cretacea under low positive temperatures. It is possible that it will be enough to maintain the culture in the medium without phytohormones during the summer dormancy. Yu et al. (2012) confirmed the latter suggestion for the culture of herbaceous Paeonia lactiflora. In our work, two waves of morphogenesis of S. cretacea were observed: the activation of axillary meristems and development of side shoots from them occurred during the first two weeks of cultivation. After this, the rapture of nodal region of explants took place, and the formation of bud-like structures from cells of the main parenchyma, which we called “callus-like tissue” on the stage of the selection of growth regulators, occurred during the third week. It is known that totipotency of inherently parenchymal meristematic cells implemented during their division and differentiation, whereas the totipotency of specialized
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 47
No. 6
2016
FEATURES OF CLONAL MICROPROPAGATION
t p
ph
the course of in vitro growth of such fasciated shoots, their division into separate shoots, which formed already normal shoots growing by one apical apex, occurred. This fact indicates that the observed effect is not associated with abnormalities in the genetic control of the development and it is due to natural physiological reasons, for example, the result of pressure of intensively proliferating tissues of nodal regions on the apexes. ACKNOWLEDGMENTS
c
m
The study was supported by the Russian Ministry of Education as a part of the basic section of the government task for scientific activities according to task no. 2014/203, project code 1287.
x e
Fig. 3. Cross section of the stem of S. cretacea intact plant, stained according to D. Tolivia and J. Tolivia (Tolivia, D. and Tolivia, J., 1987): c—collenchyma; e—epidermis; m— medulla; p—parenchyma; ph—phloem; t—trichomes; x— xylem. Scale: 250 μm.
cells implemented via dedifferentiation is followed by a new differentiation (Zhuravlev and Omelko, 2008). Since parenchymal cells have relatively low differentiation, i.e., they are not morphologically and physiologically specialized and can change during ontogeny or perform several different functions at the same time, including participation in wound healing and regeneration processes (Esau, 1997), the formation of bud-like structures observed during the third week of cultivation was not associated with the processes of dedifferentiation. As can be seen in Fig. 3, S. cretacea does not have a beam type structure; i.e., the conductive system is a closed ring surrounded by a thick layer of the main parenchyma and collenchyma. In the nodal region, conducting system of the stem connected with the conducting system of the leaf and layers of mechanical tissue are less significant, which is probably why the rapture appears in this area. D.S. Kulhanova et al. (2015) detected formation of similar morphological structures. The difference is the location of de novo formed bud-like structures across the whole surface of the explant, without association with damaged areas, and their origin from the epithelial tissue. The ribbon-like fasciation observed in our experiment is the result of intergrowth of apical and axillary apexes of shoots or several diametrically opposed, coplanar axillary apexes, since it occurs also during the formation of the shoot with a similar anomaly in situ (Choob and Sinyushin, 2012). In the future, in RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
365
REFERENCES Aescht, E., Büchl-Zimmermann, S., Burmester, A., et al., Färbungen, in Romeis—Mikroskopische Technik, Mulisch, M. and Welsch, U., Eds., Springer Spektrum, 2010a, pp. 181–297. doi 10.1007/978-3-8274-2254-5 Aescht, E., Büchl-Zimmermann, S., Burmester, A., et al., Präparationstechniken und Färbungen von Pflanzengewebe für die Lichtmikroskopie, in Romeis— Mikroskopische Technik, Mulisch, M. and Welsch, U., Eds., Springer Spektrum, 2010b, pp. 317–338. doi 10.1007/978-3-8274-2254-5 Báthori, M., Tóth, N., Hunyadi, A., et al., Phytoecdysteroids and anabolic-androgenic steroids—structure and effects on humans, Curr. Medicinal. Chem., 2008, vol. 15, no. 1, pp. 75–91. doi 10.2174/092986708783330674 Bilz, M., Kell, S.P., Maxted, N., et al., European Red List of Vascular Plants, Luxembourg: Publications Office of the European Union, 2011. Cheng, D.M., Yousef, G.G., Grace, M.H., et al., In vitro production of metabolism-enhancing phytoecdysteroids from Ajuga turkestanica, Plant Cell Tiss. Org. Cult., 2008, vol. 93, no. 1, pp. 73–83. doi 10.1007/ s11240-008-9345-5 Choob, V.V. and Sinyushin, A.A., Flower and shoot fasciation: from phenomenology to the construction of models of apical meristem transformations, Russ. J. Plant Physiol., 2012, vol. 59, no. 4, pp. 530–545. doi 10.1134/ S1021443712040048 Devyatov, A.G., Cretaceous catchfly Silene cretacea Fisch. ex Spreng, in Krasnaya kniga Rossiiskoi federatsii (rasteniya i griby) (The Red Data Book of the Russian Federation (Plants and Fungi)), Moscow: Tovar. nauch. izd. KMK, 2008, pp. 172–173. Esau, K., Anatomy of Seed Plants, 2nd ed., New York: Wiley, 1997. George, E.F., Hall, M.A., and De Klerk, G.J., Plant growth regulators III: gibberellins, ethylene, abscisic acid, their analogues and inhibitors; miscellaneous compounds, in Plant Propagation by Tissue Culture, vol. 1: The Background, 3rd ed., George, E.F., Hall, M.A., and Klerk, G.J., Eds., Dordrecht: Springer Netherlands, 2008, pp. 227– 281. doi 10.1007/978-1-4020-5005-3_7 Vol. 47
No. 6
2016
366
KRITSKAYA et al.
Kritskaya, T.A. and Kashin, A.S., Using in vitro culture methods for the conservation of some rare and endangered species of calciphilic plants of Saratov oblast, Izv. Saratov. Univ., Nov. Ser., Ser. Khim. Biol. Ekol., 2013, vol. 13, no. 4, pp. 65–73. Kritskaya, T.A., Blyudneva, E.A., and Kashin, A.S., The culture medium for micropropagation of calciphilic plants in culture in vitro, RF patent no. 2552174 C1, Byull. Izobret., 2015, no. 16. Kul’khanova, D.S., Erst, A.A., and Novikova, T.I., In vitro regeneration from bulbous scales of Fritillaria sonnikovae, an endemic species, Russ. J. Dev. Biol., 2015, vol. 46, no. 4, pp. 215–221. Libbert, E., Lehrbuch der Pflanzenphysiologie, 5th ed., Jena: Gustav Fisher Verlag, 1993. McCown, B.H. and Lloyd, G., Woody plant medium (WPM)—a revised mineral nutrient formulation for microculture of woody plant species, Hort. Sci., 1981, vol. 16, p. 453. Mitrofanova, I.V., Somaticheskii embriogenez i organogenez kak osnova biotekhnologii polucheniya i sokhraneniya mnogoletnikh sadovykh kul’tur (Somatic Embryogenesis and Organogenesis as a Basis for Biotechnology of Obtaining and Maintaining Perennial Horticultural Crops), Kyiv: Agrarna nauka, 2011. Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant., 1962, vol. 15, no. 13, pp. 473–497. Nevskii, S.A., Davidenko, O.N., Berezutskii, M.A., et al., On the finding of cretaceous cachfly (Silene cretacea Fisch. ex Spreng., Caryophyllaceae) in Saratov oblast, Povolzhsk. Ekol. Zh., 2009, no. 2, pp. 170–172. Polevoi, V.V. and Salamatova, T.S., Fiziologiya rosta i razvitiya rastenii (Physiology of Plant Growth and Development), Leningrad: Izd. Leningr. Univ., 1991. Ponert, J., Vosolsobě, S., and Kmecová, K., et al., European orchid cultivation—from seed to mature plant, Eur. J. Environ. Sci., 2011, vol. 1, no. 2, pp. 95–107. Riordan, J., An Introduction to Combinatorial Analysis, Mineola, New York: Dover Publ., 2002. Skoog, F. and Miller, C.O., Chemical regulation of growth and organ formation in plant tissues cultured in vitro, Symp. Soc. Exp. Biol., 1957, vol. 11, pp. 118–131.
Spivak, V.A., Morphogenetic variability in the apical bud of wheat shoot and its dependence on the light factor in the culture in vitro, Extended Abstract of Cand. Sci. (Biol.) Dissertation, Moscow: MGU im. M.V. Lomonosova, 1994. Van Staden, J., Zazimalova, E., and George, E.F., Plant Growth Regulators II: Cytokinins, their analogues and antagonists, in Plant Propagation by Tissue Culture, vol. 1: The Background, 3rd ed., George, E.F., Hall, M.A., and Klerk, G.J., Eds., Dordrecht: Springer Netherlands, 2008, pp. 205–226. doi 10.1007/978-1-4020-5005-3_6 Tolivia, D. and Tolivia, J., Fasga: a new polychromatic method for simultaneous and differential staining of plant tissues, J. Microsc., 1987, vol. 148, no. 1, pp. 113– 117. doi 10.1111/j.1365-2818.1987.tb02859.x Tsvelev, N.N., Genus Silene L, in Flora Vostochnoi Evropy (Flora of Eastern Europe), vol. 11: Pokrytosemennye (Angiosperms), Moscow: Tovar. Nauch. Izd. KMK, 2004, pp. 233–247. Tuleuov, B.I., Turdybekov, K.M., Khabdolda, G., et al., Structure and stereochemistry of phytoecdysone from Silene cretacea Fisch., Russ. J. Gen. Chem., 2014, vol. 84, no. 4, pp. 704–707. doi 10.1134/S1070363214040173 Vetchinkina, E.M., Shirnina, I.V., Shirnin, S.Yu., et al., Conservation of rare species of plants in genetic collections in vitro, Vestn. Balt. Gos. Univ. im. I. Kanta, 2012, no. 7, pp. 109–118. Yu, X., Wu, H., Teixeira da Silva, J.A., et al., Multiple shoot induction and rooting of Paeonia lactiflora ‘Da Fu Gui’, Afr. J. Biotechnol., 2012, vol. 11, no. 41, pp. 9776– 9781. Zholobova, O.O., Conservation of rare and endangered plant species in in vitro culture and estimation of the level of their intraspecific polymorphism, Extended Abstract of Cand. Sci. (Biol.) Dissertation, Belgorod, 2012. Zhuravlev, Yu.N. and Omelko, A.M., Plant morphogenesis in vitro, Russ. J. Plant. Physiol., 2008, vol. 55, no. 5, pp. 579–596. doi 10.1134/S1021443708050014
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Translated by V. Mittova
Vol. 47
No. 6
2016
