Rare and endemic plant species of Mountain Crimea flora: a morphogenic response to conservation in the gene bank in vitro

light intensity, increasing the osmotic concentration, introducing retardants into the culture medium. For conservation, we used microshoot segments of Lamium glaberrimum (K. Koch) Taliev (Lamiaceae), Crepis purpurea L. (Asteraceae), Scrophularia exilis Popl. (Scrophulariaceae), Silene jailensis N. I. Rubtzov (Caryophyllaceae) cultured under standard in vitro conditions for 8‒10 months. The explants were placed on ¼ MS culture medium supplemented with plant growth inhibitors: 0.2 g/L 2-chloroethyl-trimethyl-ammonium chloride (CCC) and 60 g/L sucrose. Conservation conditions were: the temperature 4–6 °C, light intensity 1.25–3.75 µM m -2 s -1 , photoperiod 16 hours. Screening of the morphometric pa rameters indicated high viability and a decrease in growth kinetics after 12 and 24 months of deposition in the gene bank in vitro . Along with the microshoot slow growth, the formation of adventitious shoots, leaves, and roots was observed.


Introduction
Nowadays, an important part of the world biodiversity conservation is the possibility of plant resources depositing in vitro, which makes it possible to preserve long-time genetic collections of wild and cultivated plant species. Biotechnological approaches have been actively used in recent years to preserve rare, endemic, and endangered plant species listed in the republican Red Data Books and the Red Data Book of the Russian Federation under conditions of slow growth. This is reached by lowering the temperature and light intensity, increasing the osmotic concentration, and introducing retardants into the culture medium (Belletal, 2002;Engelmann, 2011). Rare endemic species require particular attention, since in nature their number constantly decrease due to the low degree of reproduction in nature growth conditions (Nikiforov et al., 2016;Whitlock et al., 2016;Nikiforov, 2018). Many endemic species are listed in the "Red Data Books of the Russian Federation (plants and fungi)" (2008) and "Red Book of the Republic of the Crimea (plants, algae and mushrooms)" (2015). Biotechnological methods make it possible to preserve valuable rare species, as well as single specimens in vitro, which are an integral part of the Global Plant Biodiversity Preservation concept (Sarasan et al., 2006;Cruz-Cruz et al., 2013;Engelmann, 2013;Novikova, 2013;Molkanova et al., 2018). One of the most promising ways for plant biodiversity conservation is the formation of gene banks in vitro. Nowadays, the Laboratory of Plant Biotechnology and Virology of the Nikita Botanical Gardens carries out complex studies of rare and endemic species of Mountain Crimea flora in the field of somatic embryogenesis and organogenesis induction, clonal micropropagation and subsequent long-term conservation in vitro at low positive temperatures (Mitrofanova et al., 2020). In the process of plant deposition protocols development considering their genotype, along with exposure to low positive temperatures, a complex of osmotics and retardants were used, which slow down the growth of the studied plants under in vitro conditions (Mitrofanova, 2018).
Thus, plants, maintained under different culture conditions, retain both characteristics of their genotype and acquire some new features correspond with the conditions of cultivation and conservation. Thus, specimens preserved for a long time under in vitro conditions can have various morphological changes in microshoots and leaves, structural features of organs, which enable to determine the limits of the anatomical and physiological changes, reveal the dependence of the growth processes on the studied regenerants structure (Mitrofanova, 2018).
The objective of this study was to investigate the features of long-term conservation and identify morphological and anatomical features of some rare and endemic plant species after 24 months of deposition in the genebank in vitro. Reagents 1. Macro-and microelements, vitamins were used according to ¼ МS (Murashige, Skoog, 1962 7. BIOTRON`s phytocapsules with a cool white light of fluorescent lamps (PhilipsTL, Japan).
The pH of the medium was adjusted to 5.7 before autoclaving (120 °C 10 min).
3. After 12 and 24 months conservation, the morphometric indexes of the objects were screened.

Morphometric parameters of explants maintained in vitro for 12 months
1. Morphometric parameters screening of the explants after 12 months deposition revealed that their viability on ¼ MS culture medium supplemented with 0.2 g/L CCC and 60 g/L sucrose at 4 °C was 98 % in C. purpurea plants, 95 % in S. exilis, S. jailensis and L. glaberrimum (Table).  (2022) 2. A slight growth of microshoots, the formation of green leaves and one-three adventitious microshoots per explant were noted.
3. Microshoot thickness in the studied species was 936-1230 µm, S. jailensis plants had the thinnest shoots. On the transverse sections of the microshoots, xylem and phloem vessels were visible (Fig. 1); in S. jailensis, sclerenchyma was noted.
4. The high capacity for the subsequent development of plants can be estimated by the formed axillary buds (Fig. 1.3).

Morphometric parameters of explants preserved in vitro for 24 months
1. After 24 months deposition under conditions of gene bank in vitro at a temperature 4 and 6 °C on MS culture medium supplemented with 60.0 g/L sucrose and 0.2 g/L CCC explants had different viability degree, produced single leaves, adventitious buds, shoots, rosettes, and roots. At the same time, a decrease in the growth kinetics by 1.5-3 times compared to the control was noted.
3. L. glaberrimum explants, on average, formed 2.38 ± 0.18 leaves. Their thickness was the same as after 12 months of in vitro deposition. The leaf became a little thicker, large intercellular spaces appeared in the tissues. The newly formed buds developed slowly: leaf primordia were visible (Fig.  2). At the same time, some leaves and shoots had the signs of drying out. The viability was 75 %.
4. S. exilis microshoots were 1.97 ± 0.02 cm long, 1002 µm diameter, the number of lateral shoots was 2.5 ± 0.32, and the total number of leaves was 8.25 ± 0.45. Under long-term storage, the leaf blades became thinner (248 ± 42 µm) and had an eightnine layer differentiated mesophyll. The color of the shoots varied from green to anthocyanin, the leaves -from green to green-brown. The shoots had elongated internodes. All explants had no roots. Explants viability was 85 %.
5. S. jailensis microshoots after 24 months of direct culture at a temperature 4 °C had a diameter 1600-1800 μm, were 2.9 ± 0.04 cm high, retained their viability (70 %), formed adventitious shoots of 0.63 ± 0.18 ones per shoot and 11.25 ± 0.45 leaves per shoot. Green leaves remained only at the top of the shoot. New buds formed in the axils of leaves. It is typical for the development of this species under standard culture conditions on a culture medium with plant growth regulators. Under long-term storage, the leaf blades became thinner (258-312 µm) and had an eight-nine layer mesophyll.

Conclusion
The main indicator of the successful conservation of plant species, cultivars and forms is the preservation of their physiological stability (viability) during the entire storage period with a decrease in the growth kinetics. Previous studies made it possible to determine the optimal temperatures (4-6 °C) for conservation of some rare endemics of the Mountainous Crimea. At these temperatures, the explants remained viable with minimal growth (Mitrofanova et al., 2020). The complex use of a number of factors such as sucrose and CCC in the culture medium, optimal temperature and light intensity enabled not only to prolonge the deposition time, but also to keep the viability of the studied cultures explants.
Our experiments have demonstrated morphological and anatomical stability in explants of L. glaberrimum, C. purpurea, S. exilis, S. jailensis on a culture medium supplemented with osmosis and retardants during 24 months of conservation in the gene bank in vitro at temperature -4 and 6 °C. In all studied plant species, a decrease in viability was noted with an increase in the time of deposition from 12 to 24 months. Along with the retarded shoot growth, the formation of adventitious shoots, leaves, and roots was observed. These characteristics were noted in a number of scientific works on other cultures (Kovalchuk et al., 2009;Gianni, Sottile, 2015;Rajasekharan, Shijram, 2015;Reed, DeNoma, 2016). The formation of multiple axillary buds may indicate a high regeneration capacity in rare and endemic species. A number of histological features in the leaves of the in vitro deposited explants were revealed. These features enable explants to maintain physiological stability when active production processes are stopped. In leaf blades, the thickness and density of the mesophile decreased, the polysad tissue degraded, and a high rate of transpiration regulation was noted. It was found that under conditions of long-term preservation of explants at an optimal temperature, tissues of regenerants have high viability due to the structural rearrangements of chlorenchyma and keeping a number of characteristics of each plant species under in vitro conditions.