Ge\\ Nature YS) Conservation Nature Conservation 58: 289-309 (2025) DOI: 10.3897/natureconservation.58.150920 Research Article Advances in conservation physiology and ex situ propagation strategies for the rare moss Podperaea krylovii (Amblystegiaceae, Bryophyta) Bojana Z. Jadranin™®, Marija V. Cosié’®, Djordje P. Bozovic'®, Milorad M. Vujicié'®, Michael S. Ignatov?*®, Aleksey V. Troitsky®®, Aneta D. Sabovljevic'?®, Marko S. Sabovljevic!?°© Oo oO FP WO DY Institute of Botany and Botanical garden “Jevremovac’, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia Center of Plant Biotechnology and Conservation (CPBC), Takovska 43, 11000 Belgrade, Serbia Tsitsin Main Botanical Garden, Russian Academy of Sciences, Botanicheskaya Str. 4, Moscow 127276, Russia Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory Str. 1-12, Moscow 119234, Russia Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia Department of Plant Biology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Safarik University in Kosice, Manesova 23, 040 01 Kosice, Slovakia Corresponding author: Marko S. Sabovljevié (marko@bio.bg.ac.rs) OPEN Gaccess Academic editor: Yuanjun Zhu Received: 20 February 2025 Accepted: 22 April 2025 Published: 20 May 2025 ZooBank: https://zoobank. org/78BCDDE1-D7E5-4FAA-9669- 0738C01E7B95 Citation: Jadranin BZ, Cosié MV, Bozovic DP, Vujicic MM, Ignatov MS, Troitsky AV, Sabovljevic AD, Sabovljevic MS (2025) Advances in conservation physiology and ex situ propagation Strategies for the rare moss Podperaea krylovii (Amblystegiaceae, Bryophyta). Nature Conservation 58: 289-309. https://doi.org/10.3897/ natureconservation.58.150920 Copyright: © Bojana Z. Jadranin et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract This study investigates the growth and micropropagation of the rare moss Podperaea krylovii, under in vitro conditions. The effects of different growth media, plant growth regulators, and sugars were tested to optimize conditions for efficient propagation and to gain a better understanding of the species’ biology and the cultivation requirements of this species. The results indicate that the KNOP medium is most effective for the propagation of P krylovii, with prostrate-oriented explants having a higher index of multi- plication compared to upright-oriented explants. The addition of auxin had no significant effect on species multiplication and development in comparison to auxin-free media. Conversely, cytokinin inhibited shoot formation as well as protonemal development, suggesting that its use individually was not suitable for the species’ development under axenic conditions. The sugars tested showed no positive effect on new shoot formation, suggesting that no exogenous carbon source is required and that this species keeps autotrophy in axenic cultures. These results provide a basis for future research on the propagation and cultivation of P krylovii and emphasize the need for specific cultivation protocols. The in vitro multiplicated moss was successfully acclimatised and propagat- ed ex vitro in a xenic environment. This enables further successful ex situ conservation of the species as well as commercial use as aquatic plants, for example in aquaria. Key words: Acclimation, axenic culture, bryophytes, micropropagation, plant growth regulators, Sugars Introduction The amblystegian pleurocarp moss Podperaea krylovii (Podp.) Z. lwats. & Glime (synonym Chrysohypnum krylovii Podp.; syn. Campylium krylovii (Podp.) Laz.) was initially described as a genus and species new to science, belonging to the family Hypnaceae (Iwatsuki and Glime 1984). However, later molecular studies showed 289 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii that this species is more closely related to members of the family Amblystegia- ceae (Ignatov and Milyutina 2011). The species is rare, but morphologically and molecularly confirmed as a good taxon. Although it is also easily recognized in the field by its superficial resemblance to Campylium sommerfeltii Myr., but easily distinguished by the double-celled teeth on the leaf margin, not so many ecological and biological features are known for this species due to its rarity and the rather small number of records. The currently known distribution area includes south Si- beria (westward to Altai), the Russian Far East, northern Japan (Hokkaido), and northeastern China (Ignatov and Afonina 1992; Ignatov et al. 1996; Ignatov and Mi- lyutina 2011). The records from the Inner Mongolia autonomous District in China led to the description of the second species of this genus, namely P. baii Ignatov (both morphologically and molecularly confirmed) (Ignatov and Milyutina 2011). The biology of the species is rather poorly known and due to its rarity, very little can be found in the literature about this species. For conservation purpos- es and fundamental issues, such species are of high priority both for biological feature studies (e.g. Rincon 1993; He et al. 2016; Pakeman et al. 2019; Calleja et al. 2022) and biotechnology investigations (e.g. Gonzalez et al. 2006; Mallon et al. 2007; Decker and Reski 2020). Even in the original description (Ilwatsuki and Glime 1984) the ecological characteristics are not given. It inhabits a variety of habitats, including eroded soils on slopes in open places, areas near roads, sites under loose canopy in deciduous forests, soils at the base of cliffs, rock out- crops in non-dense forests, soils above fallen logs in valleys, and also slopes by ponds (Iwatsuki and Glime 1984; Ignatov et al. 2022). The same author gave the altitude of occurrence as 70-450 m, rarely up to 900 m. The species is known to be autoicous (Iwatsuki and Glime 1984) and to produce sporophytes relatively frequently. It seems that the species does not remain in the same location for long, as repeated visits to the same site often do not confirm the presence of the species (pers. comm. Ignatov). The species is regarded as data deficient (DD) under the IUCN criteria. However, its survival may be considered under threat due to habitat loss and sensitivity to environmental and climatic changes. This study aims to document the biology and developmental features of the species, bearing in mind that this species is rarely empirically documented in remote and uninhabited areas of North Asia. These can be successfully used in the action plans for ex situ conservation of the rare moss P. krylovii by optimiz- ing axenic and xenic ex situ propagation if needed. Indeed, bryophyte conserva- tion can be assessed as neglected in comparison to tracheophytes, and bear- ing in mind that this group is very heterogeneous and less known, it is not easy to apply biotechnics and biotechnology known to be used in vascular plants. In this work, we addressed the following key questions: (1) What is the opti- mal explant orientation and growth medium type for micropropagation of the species under axenic conditions? (2) What are the effects of the exogenous addition of selected plant growth regulators (PGRs) to the growth medium on the morphogenesis of the species and can explant orientation modify their ef- fects? (3) How do exogenously added different sugars (carbon sources and signaling compounds) modify morphogenesis and can they enhance the prop- agation of the species? (4) Can leafy gametophores be used for successful acclimation and xenic propagation ex situ and/or commercially as aquarium plants, e.g. as a conservation method? Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 290 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii Methods Plant material and in vitro culture establishment The plant material used in this study was obtained from specimens grown un- der terrarium conditions at Lomonosov University in Moscow by A. Troitsky (August 02, 2013), and previously collected in Primorsky Territory, Russia. This specimen is deposited in the MHA (Herbarium of the Main Botanical Garden of the Russian Academy of Sciences), Russia, as voucher Ignatov #07-220 and was also used for sequencing [Gen-bank #JN896329]. This accession was used for in vitro (axenic) experimentations, while moss material from, Lozovyi Range, Partizansk Distr. (Russia), voucher MHA901589, September 08, 2013, Ignatov M.S., Ignatova E.A., Malashkina E.V., #13-1856 (S-facing slope, open oak stand with Lespedeza bicolor Turcz., 43°00'N, 133°00'E) was used for xenic propagation of plant material. Surface sterilization of both gametophytes and sporophytes was performed with different concentrations of ethanol (50% and 90%), sodium hypochlorite (NaOCl) (1%, 3%, 5%, and 7%), and sodium dichloroisocyanurate (NaDCC) (1%, 3%, 5%, and 7%) for different time periods (1, 3, 5, and 7 minutes). While the other solutions tested were lethal to both the gametophytes and spores or were ineffective in eliminating contamination by xenic organisms and contaminants, the 3% NaDCC solution used for 5 minutes proved to be the most effective solution for sterilizing the plant material. Once the axenic culture of P krylovii was established, individual 1 cm long and decapitated explants were used for the experiments, which included differ- ent media types with or without supplementation (e.g. plant growth regulators or sugars). Decapitation (removing the tip of the plantlets) eliminates divid- ing cells and minimizes possible errors in the measurement of regeneration and morphological parameters. This ensures that each explant has an equal chance to de-differentiate and initiate the formation of a new meristem and tips. The explants were placed in different treatments on the media whose pH was adjusted to 5.8 before being autoclaved at 121 °C for 30 minutes. All treat- ments used in this study consisted of four individual explants in five separate Petri dishes, i.e. a total of 20 gametophores per treatment. The explants were cultured under axenic conditions in sterile Petri dishes at a temperature of 18 + 2 °C and a humidity of 60-70% and exposed to a long-day light cycle (16 h light/8 h dark) with fluorescent tubes (Tesla Panéevo) providing a light intensity of 50 umol ms“! Photosynthetic Photon Flux Density (PPFD). Experimental design This study was conducted in three different experimental setups (see Fig. 1). The “Medium type experiment” focused on examining the effects of different growth media on the growth and development of the moss P krylovii. Explants were placed on solid KNOP medium (Knop 1865), solid half-strength Murashige & Skoog medium (MS/2) (Murashige and Skoog 1962; Cosié et al. 2025a), and solid BCD medium (Cosié et al. 2025a). The plantlets were positioned both up- right and prostrate on the media to investigate whether the position of the ex- plants influences morphogenesis. Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58. 150920 291 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii _ Medium type experiment _ PGR experiment _ tl Cc oO. i — oO jos oO oO jos > pete m4 Oo oH =a nO Figure 1. A diagram of the experimental design. The “Medium type experiment” included three different solid media types (KNOP MS/2 and BCD) on which the explants were placed in two positions, either upright or prostrate. The “PGR experiment” included ex- perimental setups with different concentrations (0.03, 0.3, and 3 uM) of IBA and BAP added to the KNOP medium on which the explants were placed in two positions, either upright or prostrate. In the “Sugar type experiment”, different concentrations (0.04 and 0.08 M) of four sugars (fructose, glucose, maltose and sucrose) were added to the KNOP medium on which the explants were placed exclusively in upright position. In all experiments performed, each treatment consisted of four explants in five Petri dishes (a total of 20 explants per treatment). The “PGR experiment” investigated the effect of plant growth regulators (PGRs), in particular auxins and cytokinins, on the growth and development of the target moss. Different concentrations (0.03, 0.3, and 3 uM) of IBA (in- dole-3-butyric acid) and BAP (6-benzylaminopurine) were applied exogenously to the KNOP medium on which the explants were placed in both upright and prostrate positions. The KNOP medium was chosen because the gameto- phores developed well and formed numerous new shoots without protonema as inferred from the results of the “Medium type experiment”. In the “Sugar type experiment”, different concentrations (0.04 and 0.08 M) of four sugars were added individually to the KNOP minimal medium with aim to document its action as signal molecules and/or source of carbon in further development. The primary sugar concentration used in this experiment was Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 299 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii set to half-strength (15 g/L) compared to the concentration commonly used in MS medium (30 g/L) for all four sugars, as bryophytes do not require high con- centrations of carbon sources for their growth in vitro. Since the half-strength molar concentration is 0.04 M for maltose and sucrose and 0.08 M for fructose and glucose, we used both concentrations for all tested sugars to enable a comparative analysis of the results. After 4 weeks, the morphogenetic changes were observed, including the for- mation of new shoots on the initial explant (i.e. index of multiplication), the diameter of the secondary protonema and the survival rate. The morphological parameters were measured and photographed using a Leica MZ stereomicro- scope (Leica MZ 7.5, Bi-Optic Inc., Santa Clara, CA, USA). Acclimation of in vitro propagated Podperaea krylovii The optimization of in vitro propagation enabled sufficient biomass produc- tion and acclimation of the investigated species. The plantlets were transferred from axenic in vitro to controlled xenic laboratory conditions and acclimation lasted for four weeks. The moss material was placed in Magenta containers filled with distilled water on white terrazzo marble gravel chippings (5-8 mm in size). The environmental conditions were at a temperature of 18 + 2 °C, rel- ative humidity of 100%, and continuous light with a 24-hour photoperiod. The light source consisted of fluorescent tubes (Phillips Lighting, Amsterdam, The Netherlands) with a flux density of 35 umol m? s". Additionally, translocation test to controlled conditions in a growth chamber (Sanyo Environmental Test Chamber MLR-352H) on the natural substrate to xenically propagate the material. Statistical analysis The entire statistical analysis was conducted using the R programming language (v. 4.3.2) (R Core Team 2023). The first step of the analysis consisted of prelim- inary data exploration using the Shapiro-Wilk normality test and Levene test for homogeneity of variance, revealing that normality and homoscedasticity were violated across the groups. Consequently, for all of the measured parameters in all of the experiments, a nonparametric factorial ANOVA was performed em- ploying the Align Rank Transform (ART) procedure (Wobbrock et al. 2011; Elkin et al. 2021) from “ARTool” R package (Kay et al. 2021). The factorial models were created using the “art” function and the significance of the main effects and in- teractions was evaluated with the “anova” function, after which the contrast test was performed using the “art.con” function of the mentioned R package. Results The effects of explant position and growth medium type on the morphogenesis of Podperaea krylovii In the initial experiment (“Medium type experiment”), the main effects of explant orientation (EO) and medium type (MT) significantly affected both of the morphogenesis parameters i.e. the index of multiplication (IM) and Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 293 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii the diameter of secondary protonema patch (DP) (P < 0.01 and P < 0.001, respectively) in tested species P krylovii (Table 1). Furthermore, a signifi- cant interaction of the two factors (EO x MT) was observed for the DP (P < 0.001), but not for the IM (Table 1), suggesting that the explant orientation modifies the effect of the different medium types on the development of the secondary protonema. The highest IM was observed in plants grown on KNOP medium in a prostrate explant orientation, followed by plants on the same medium in an upright orientation, while the lowest IM values were documented in plants grown on MS/2 medium (Fig. 2A). Moreover, a clear distinction among the three tested media types was evident, with all showing significantly differ- ent IM values in both prostrate and upright explant orientations (P < 0.05). However, no significant differences were observed between the two orienta- tions within any of the media types (Fig. 2A). Interestingly, for all tested me- dia types, plants grown in an upright orientation exhibited lower IM values compared to their prostrate counterparts although these differences were not statistically significant (Fig. 2A). Concerning the secondary protonema development, the highest DP values were observed in plants grown on BCD medium in an upright orientation, with all other DP values for any combination of explant orientation and medium type being significantly lower (P < 0.05) (Fig. 2B). Interestingly, plants grown on KNOP medium in a prostrate explant orientation did not develop any secondary protonema (Fig. 2B). This unique response, together with the highest IM values for this combination of explant orientation and medium type (prostrate orienta- tion of explants grown on KNOP medium) suggest this combination is optimal for biomass production of P krylovii. Explants grown on KNOP and BCD media in both prostrate and upright orientation formed a remarkable number of gametophores (Fig. 3A, B, D, E) compared to those grown on MS/2 medium (Fig. 3C, F). On the other hand, secondary protonema developed mainly in plants positioned upright on the media (Fig. 3D—F). However, when placed prostrate, plants grown on BCD and MS/2 media formed green secondary protonema, but to a lesser extent than the upright-oriented explants. Of all the media tested, the KNOP medium was suitable for the formation of massive green gametophores when the explants were positioned prostrate (Fig. 3B), and was therefore selected for further ex- periments. Plants grown on MS/2 medium did not develop new gametophores, although secondary protonema was documented, especially when the plants were positioned upright (Fig. 3C, F). Table 1. Summary of factorial analysis for “Medium type experiment”, evaluating the ef- fects of explant position (EO), medium type (MT), and their interaction (EO x MT) on the index of multiplication (IM) and the diameter of the secondary protonema patch (DP) in P krylovii. The values represent F-statistics, with asterisks indicating levels of statistical significance (**P < 0.01, ***P < 0.001). Parameter EO MT EO x MT IM 7.3527%** 51.091 5*** 1.7222 DP 24.681 5*** 5.2304** 12.2429%*** Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 294 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii A B Cc F 4, = 20 on L © lok © 5 Ee = 34 © 1.5 = g 2 ab e = fo¥ - S24 3 1.0 Pas re se) ® £ 77) yu ro) L£ oO = ay Qa 0-4 0.0 BCD KNOP MSs/2 BCD KNOP MS/2 Growth medium Growth medium Explant orientation Ml prostrate MEM upright Figure 2. The effects of explant position and different growth media types on the index of multiplication (A) and the diameter of secondary protonema patch (B) were assessed in the “Medium type experiment”. Data are presented as mean + standard error. The letters above the bars represent statistically significant differences (P < 0.05) among the experimental groups. BCD KNOP MS/2 Prostrate Upright Figure 3. The appearance of Podperaea krylovii explants grown on different media types. The effects of explant position and exogenous plant growth regulators on the morphogenesis of Podperaea krylovii In the “PGR experiment”, the effects of explant orientation (EO) and concentration (C) on morphogenesis parameters varied between IBA- and BAP-treated plants. In IBA-treated plants, explant orientation significantly affected both morphogenesis parameters evaluated (P < 0.01 and P < 0.001 for IM and DP respectively), while Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 295 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii the effect of IBA concentration was significant only for DP (P < 0.001) (Table 2). Furthermore, a significant interaction between EO and concentration (EO x C) was observed for DP (P < 0.001), suggesting that the impact of IBA concentration on secondary protonema development is modified by explant orientation. How- ever, no significance for such an interaction was found for IM. On the other hand, in BAP-treated plants, the index of multiplication was sig- nificantly affected by BAP concentration (P < 0.001), but not by explant orien- tation (Table 2). Additionally, the diameter of the secondary protonema patch was significantly affected by EO, C and their interaction (EO x C) (P < 0.001) indicating a complex relationship between secondary protonema development, explant orientation, and BAP concentration (Table 2). In the “PGR experiment’, different plant growth regulators had different impacts on the morphogenesis of P krylovii (Fig. 4). There were no statistically significant differences observed for the IM values in IBA-treated plants, although a slight, but not significant decrease can be observed in plants grown in an upright orientation compared to their prostrate counterparts for all of the applied IBA concentrations (Fig. 4A). On the other hand, IBA treatments influenced secondary protonema de- velopment differently depending on the explant orientation. In control plants with a prostrate orientation, there were no observed explants with developed secondary protonema (Fig. 4B). However, lower IBA concentrations (0.03 and 0.3 uM) strong- ly stimulated protonemal development, whereas the highest concentration (3 uM) also induced protonema formation, but resulted in significantly smaller DP values compared to the lower concentrations (P < 0.05) (Fig. 4B). The index of multiplication of BAP-treated plants with a prostrate orientation decreased with increasing BAP concentration, with all the treated plants exhibiting significantly lower IM values compared to the control group (P < 0.05) (Fig. 4C). A similar trend of IM values decreases was observed in BAP-treated upright-oriented plants. However, in this case, only those plantlets treated with the highest applied BAP concentration (3 uM) exhibited significantly lower IM values compared to the control group (P < 0.05) (Fig. 4C). This suggests that the prostrate orientation could enhance BAP absorption due to the greater surface area contact with the medium. Secondary protonema patches were not observed in control plants with a prostrate orientation (Fig. 4D). However, the lowest applied BAP concentration (0.03 uM) successfully induced secondary protonema development in these plants (Fig. 4D). On the other hand, in an upright-oriented control plant, the highest DP values were observed and interestingly, treatment with any BAP concentration completely in- hibited secondary protonema development in upright-oriented plants (Fig. 4D). Table 2. Summary of factorial analysis for “PGR experiment”, evaluating the effects of explant position (EO), concentration (C), and their interaction (EO x C) on the index of multiplication (IM) and the diameter of the secondary protonema patch (DP) in response to two plant growth regulators, IBA and BAP in P krylovii. The values represent F-statis- tics, with asterisks indicating levels of statistical significance (**P < 0.01, ***P < 0.001). PGR Parameter EO Cc EOxC IBA IM F151 313* 0.88928 0.26528 DP VE103244 9.8409*** 16.8184*** BAP IM 1.9112 19,521k**3 2.6230 DP 1962770 33:4743** 85.645*** Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 296 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii A w 1 Index of multiplication NO —_ 4 Diameter of secondary protonema patch (mm) 0) 0.03 0.3 3 0 0.03 0.3 3 IBA concentration (uM) IBA concentration (uM) oO 0 Index of multiplication Diameter of secondary protonema patch (mm) | ie ) 0.03 0.3 3 0) 0.03 0.3 3 BAP concentration (uM) BAP concentration (uM) 0- Explant orientation MMM prostrate MM upright Figure 4. The effects of explant position and varying concentrations of IBA and BAP on the index of multiplication (A — IBA and C - BAP) and the diameter of the secondary protonema patch (B — IBA and D - BAP) were assessed in “PGR experiment”. Results are presented as mean + standard error. Statistically significant differences among experimental groups are indicated by different letters above the bars (P < 0.05). The exogenously applied IBA had no effect on the formation of new gameto- phores when explants were positioned upright and prostrate (Fig. 5F). The newly developed gametophores are green, well-shaped, and similar in size to those of the control group (Fig. 3B). However, IBA promoted the formation of second- ary protonema in the prostrate-positioned explants (Fig. 5A—-C), particularly when applied as 0.3 uM (Fig. 5B), which was not observed in the control plants (Fig. 3B). At high concentrations, IBA inhibited further enlargement of secondary protonema diameter in P. krylovii (Fig. 5C). In contrast, when BAP was applied, plants developed normally in upright orientation, although failed to develop sec- ondary protonema (Fig. 5J-L). The prostrate-oriented plants were smaller and thinner compared to the control plants and to IBA-treated plants (Fig. 5G-l). Interestingly, BAP inhibited the formation of secondary protonema in all plants except when applied at the lowest concentration (0.03 uM) (Fig. 5G). Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 297 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii 0.03 uM IBA 0.3 uM IBA 3 uM IBA 2 Nd D o a = 3 o a 0.3 uM BAP ® % D e E: = D o =e Figure 5. The appearance of Podperaea krylovii explants glown on KNOP medium supplemented with different IBA (A-F) and BAP (G-L) concentrations. Control group plants are shown in the Fig. 3B, E. The effects of various sugar types on the morphogenesis of Podperaea krylovii In “Sugar type experiment” both sugar type (ST) and sugar concentration (SC) had highly significant main effects (P < 0.001) on the index of multiplication of the species (Table 3). Additionally, the interaction between sugar type and concentration (ST x SC) was also significant (P < 0.05), suggesting that the effect of sugar concentration on the index of multiplication of P krylovii varies depending on the type of applied sugar (Table 3). In the case of the diameter of secondary protonema, sugar type (ST) had a significant effect (P < 0.001), while sugar concentration (SC) did not show a significant main effect (Table 3). However, the interaction between sugar type and concentration (ST x SC) was significant (P < 0.01), implying that the effect of sugar concentration varies depending on the applied sugar type (Table 3). Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 298 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii Table 3. Summary of factorial analysis for “Sugar type experiment”, evaluating the ef- fects of various sugar types (ST), sugar concentration (SC), and their interaction (ST x SC) on the index of multiplication (IM) and the diameter of the secondary protonema patch (DP) of P. krylovii. The values represent F-statistics, with asterisks indicating levels of statistical significance (*P < 0.05, ***P < 0.001). Parameter ST Sc ST x SC IM 11.3580*** 9.5074*** 2.5797* DP 10.17142*** 0.37009 3.82904** In the “Sugar type experiment”, the exogenous addition of sugar (fructose, sucrose, glucose, and sucrose) to the KNOP medium significantly (P < 0.05) reduced the index of multiplication compared to the control group only in the case of lower applied concentrations (0.04 M) of glucose and sucrose and higher concentrations (0.08 M) of sucrose (Fig. 6A). Regarding the develop- ment of secondary protonema, different effects were observed depending on the sugar type applied. A significant increase in DP values compared to the con- trol group was observed for both applied concentrations of sucrose (P < 0.05) (Fig. 6B). In contrast, a significant decrease in DP values was observed when a lower concentration of glucose and a higher concentration of fructose were applied (P < 0.05) (Fig. 6B). The morphology of the plants treated with various sugar types was gener- ally normal (Fig. 7A—H), although developed plants were remarkably smaller compared to the control plants (Fig. 3B). Overall, glucose severely inhibited the formation of new gametophores and secondary protonema when applied at a concentration of 0.04 M (Fig. 7B, F). In all other treatments, plants developed thick secondary protonema patches. Nevertheless, the formation of new game- tophores was observed in plants grown on the KNOP medium supplemented with fructose and maltose, but those were remarkably smaller and less devel- oped than the control group (Fig. 3E). However, these differences, although present, were not statistically significant (Fig. 6A). In contrast, glucose and su- crose had negative effects on IM, especially when applied at lower concentra- tions (0.04 M) (Fig. 7B, D). Acclimation of in vitro propagated Podperaea krylovii A translocation test was carried out from native sites and native substrates in the Partizansk district (Primorsky Territory, Russia) under controlled condi- tions, as well as the test of acclimation of in vitro produced plant material to xenic aquarium growth. The plant material produced in vitro in the laboratory was tested ex situ in the laboratory (Fig. 8A, B) and also by transfer of xenic material from growth cham- bers outdoors (Fig. 8C). It was found that the plantlets grew very well when transferred to a new environment and left on the original substrate for some time. The plants were kept on loamy soil in plastic boxes that were covered to ensure high humidity and watered indiscriminately when necessary. The ex situ tests resulted in well-developed gametophytes, but the sporophytes produced appear to be morphologically rare and possibly of apogamic origin, as it has al- ready been shown in some other mosses in which low light intensity can induce Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 299 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii A B E = 2.0 cow £ © Qa © 6 E re) Cc & fe 2 re) = a E 5 ‘5 3S 1.0 Ay re} 52 o — ” 6 0.5 £ oO = & a 0.0 a) 0.04 0.08 @) 0.04 0.08 Sugar concentration (M) Sugar concentration (M) Sugars ®®! Control MMi Fructose MM Glucose ME Maltose MMMM Sucrose Figure 6. The effects of varying sugar concentrations and explant position on the index of multiplication (A) and the diameter of the secondary protonema patch (B) were assessed in the “Sugar type experiment”. Results are presented as mean + standard error, with statistically significant differences among experimental groups denoted by distinct letters above the bars (P < 0.05). Fructose Glucose Maltose Sucrose 0.04 M 0.08 M Figure 7. The appearance of Podperaea krylovii explants in upright position grown on KNOP medium supplemented with different sugar types and concentrations. Control group plants are shown in the Fig. 3E. apogamy, both in Amblystegiaceae and other unrelated groups (Sabovljevié et al. 2022 and the references therein). Numerous sporophytes of P. krylovii did not reach maturity, or produce spores and were also morphologically abnormal, suggesting apbogamous development. Under these conditions, sporophytes of- ten do not appear until 2 to 4 weeks after the start of the test. Attempts were made to acclimatize the species to the water conditions for faster growth in aquaria filled with distilled water without aeration. The tem- perature was 18 + 2 °C ata constant light condition of 35 umol m?*s" PPFD. The initial results are promising, as within four weeks the size of the plants in the water has increased many times over, and many new branches have emerged. Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 300 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii Figure 8. Aquarium acclimatization (indoor) of P. krylovii originated from in vitro (axenic) cultures (A — at the beginning, B — four weeks after); C Ex situ (xenic) propagation on native substrate (outdoor) from the material originated from na- ture with the morphologically strange shape of sporophytes, possibly of apogamous origin. Discussion Influence of growth medium type and explant position on the morphogenesis of Podperaea krylovii Given the limited literature and data available on the selected species, studying the effects of different types of growth media on the morphogenesis of P. kry- lovii will lead to a better understanding of the growth and development of the species in axenic conditions. The temperature and selection of PGR concen- trations were selected based on previous experience and to allow comparison with other species (Sabovljevié et al. 2022 and the references therein). From the results obtained, it appears that of the three types of media tested, the solid KNOP medium was the most suitable for further propagation and multi- plication; regardless of whether the plantlets were oriented prostrate or upright (Figs 2A, 3B, E). Prostrate-oriented explants grown on KNOP medium had a higher number of newly formed buds and did not develop any secondary pro- tonema (Fig. 2B), which could indicate that this orientation is particularly favor- able for P. krylovii gametophore development. Additionally, prostrate-oriented explants are fully immersed in the culture me- dium on one side, allowing them to take up elements from the substrate more effectively. In contrast to P. krylovii, upright-oriented explants of another pleu- rocarpous moss species Drepanocladus lycopodioides (Brid.) Warnst (Jadranin et al. 2024) formed a higher number of new shoots, probably due to increased airflow around the explants, nutrient uptake through the wounded ends of the explants (Papafotiou and Martini 2009), and active cell-to-cell transport (Oliver and Bewley 1984). The results obtained in this study showed that higher bio- mass production (higher index of multiplication) is achieved when the surface of the plant is in close contact with the medium so that a larger surface area is involved in nutrient uptake. Compared to the prostrate-oriented explants, an upright orientation on the KNOP medium led to the formation of both second- ary protonema and new shoots of P krylovii (Figs 2A, B, 3E). In addition, plants developed new shoots also when grown on BCD medium, especially when posi- tioned prostrate, albeit to a significantly lesser extent compared to plants grown on KNOP medium (Fig. 2A). However, the secondary protonema was smaller when explants were grown on BCD medium in a prostrate position compared to upright-oriented explants (Fig. 3A, D), which may indicate that the plant invests more resources in the development of gametophores when oriented prostrate. Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 301 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii On the contrary, MS/2 medium was not suitable for the propagation of P kry- lovii, but similar to the other two tested media types, plants placed prostrate exhibited a slightly higher index of multiplication (Fig. 2A). In this case, the pro- tonema developed in both upright- and prostrate-oriented explants (Figs 2B, 3C, F). Considering that P krylovii inhabits wetlands with distinct microclimatic conditions, it is suggested that KNOP medium is well-suited for propagating P. krylovii due to its nutrient composition, which likely aligns with the specific nutritional requirements of the species. In addition, KNOP medium is widely used for the in vitro cultivation of a variety of bryophyte species (Ahmed et al. 2010; Liu et al. 2016; Sabovljevié et al. 2022) such as mosses, liverworts (Awashti et al. 2013), and peat mosses (Sastad et al. 1998; Beike et al. 2014; Natcheva and Cronberg 2023). The BCD medium has proven to be less effec- tive compared to KNOP, probably due to its specific composition and higher nitrogen content (KNO,), which exceeds the needs of the selected species. However, with minor modifications, this medium has been shown to be suit- able for certain moss species such as Hennediella heimii (Hedw.) R.H. Zander, Molendoa hornschuchiana (Hook.) Lindb. ex Limpr., Hypnum cupressiforme Hedw. and Entosthodon hungaricus (Boros) Loeske, but it is not used as fre- quently as KNOP medium (Sabovljevic et al. 2022 and the references there- in). Interestingly, MS/2 was a suitable medium for the propagation of some species such as Pterygoneurum sibiricum Otnyukova (Jadranin et al. 2023), Eurhynchium praelongum (Hedw.) B.S.G. or also mentioned H. cupressiforme (Sabovljevié et al. 2022 and the references therein). In this study, as in the case of D. lycopoidoides (Jadranin et al. 2024) or Campyliadelphus elodes (Lindb.) Kanda (Jadranin et al. 2025), MS/2 medium was not suitable for propagation. One of the reasons for this could be that the MS/2 medium probably does not meet the nutritional requirements of P krylovii since it contains higher con- centrations of nitrate ions derived from different mineral salts. Compared to the KNOP medium which contains only Ca(NO,), x 4H,O as a nitrogen source, MS/2 is composed of two different mineral salts such as NH,NO, and KNO,. Therefore, it is suggested that additional ammonium ions can contribute to such adverse results. Although further detailed studies are necessary, these findings provide a solid basis for research on the selected species. A funda- mental understanding of its nutrient requirements and habitat conditions could be crucial for future studies. Influence of plant growth regulator and explant position on the morphogenesis of Podperaea krylovii Previous studies (Ashton et al. 1979; Bhatla and Chopra 1981; Jadranin et al. 2023) have shown that plant growth regulators (PGRs) can influence develop- ment or increase the number of newly formed shoots in bryophytes. Cytokinins and auxins are likely to play important and interdependent roles in several steps of gametophytic development in bryophytes (Chopra and Sarla 1986; Anglana et al. 2024). To investigate the effects of IBA and BAP on the morphogenetic parameters of P. krylovii, different concentrations (0.03, 0.3, and 3 uM) were added individually to the basal KNOP medium on which the plant explants were placed both upright and prostrate. Although exogenously applied IBA led to the formation of new buds, none of the applied IBA concentrations resulted in a Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 302 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii higher number of newly formed buds (Fig. 4A) compared to the control treat- ment. These results indicate that KNOP medium without IBA addition is the most suitable for the propagation of P krylovii, i.e. that this species forms alarge number of new shoots regardless of the presence of PGRs. Similar findings were observed for Physcomitrella patens (Hedw.) Bruch & Schimp., which can spontaneously form a large number of new buds (von Schwartzenberg 2009). Exogenous application of auxins at times can disturb the endogenous level of these hormones and generally inhibit bud formation in mosses, particularly when used in higher concentrations (Sarla and Chopra 1987). It was previously found that in the case of decapitated moss Plagiomnium cuspidatum (Hedw.) T. Kop. exogenous auxins inhibited the formation of new buds on primary shoots (Nyman and Cutter 1981). However, in this study, none of the used IBA con- centrations were inhibitory for the formation of new gametophores. For all IBA concentrations tested, prostrate explants had a higher index of multiplication than upright explants (Fig. 4A). This result also supports the assumption that the prostrate orientation enables efficient uptake of nutrients and hormones from the medium by P krylovii. In contrast to the studies done on D. lycopoidoi- des (Jadranin et al. 2024), Entosthodon pulchellus (H. Philib.) Brugués (Cosié et al. 2025b), and P. sibiricum (Jadranin et al. 2023), where IBA led to a significant decrease in the formation of new shoots, such a result was not observed in P krylovii. Regarding the effects of IBA on the formation of secondary protonema, the results show that protonemal development occurs in prostrate-oriented ex- plants, except at the highest IBA concentration where protonemal growth was partially inhibited. On the contrary, in upright-oriented explants, the diameter of the secondary protonemata decreased upon IBA treatment (Fig. 4B). Compara- ble results were obtained for certain mosses such as P. sibiricum (Jadranin et al. 2023), E. pulchellus (Cosié et al. 2025b), and Hennediella heimii (Sabovljevic et al. 2022 and the references therein) where PGR concentrations also led to a reduction in the diameter of secondary protonemata. Influence of different types of sugars on the morphogenesis of Podperaea krylovii Plant responses to sugars, both as energy sources and signaling molecules can differ among bryophyte species. Therefore, it would be beneficial to study different types of sugar to determine how the selected species will respond to their exogenous addition. Four types of sugar were tested at two different con- centrations (0.04 and 0.08 M) to examine the response of P krylovii to specific sugars. These sugars were incorporated into the basal KNOP medium, which had been found to be the most suitable medium in prior testing. According to the results obtained, only a lower concentration of glucose and both sucrose concentrations reduced the number of newly formed shoots (Fig. 6A, 7B, D, H). Glucose also reduced the diameter of secondary protonema while sucrose in- creased it. Similar results were obtained in a study on D. lycopodioides with the same types of sugars (Jadranin et al. 2024), suggesting that these species do not require additional sugars as an additional carbon source to increase their biomass. This can be explained by the fact that bryophytes in axenic culture, when exposed to light, actively grow with high photosynthetic activity (Katoh 1983; Bozovi¢ et al. 2024), eliminating the need for an additional energy source. Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 303 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii Comparable results were observed in Atrichum undulatum (Hedw.) P. Beauv. where the applied sugars had an inhibitory effect on the development of new buds and also negatively affected the development of secondary protonema (Sabovijevié et al. 2022 and the references therein). Pterygoneurum sibiricum exhibited equivalent results, with the highest number of new shoots forming when grown on basal KNOP medium, while the addition of sugar inhibited shoot formation (Jadranin et al. 2023). On the other hand, certain studies have reported that exogenously added sugars can positively impact the growth of particular species, highlighting species-specific differences in sugar utilization and physiological responses. Such results were observed in Bryum argenteum Hedw. (Sabovljevié et al. 2022 and the references therein), where exogenously added sugars led to an increase in newly formed shoots. In addition, Pogona- tum urnigerum (Hedw.) P. Beauv. and Dicranum scoparium Hedw. are species whose growth was positively influenced by the addition of sugars in the growth medium (Sabovljevié et al. 2022 and the references therein). Experiments con- ducted on Funaria hygrometrica Hedw. and Leptobryum pyriforme (Hedw.) Wil- son with sugars such as glucose, sucrose, maltose and fructose have shown that these species can grow in the dark when one of these carbon sources is available (Simola 1969), indicating the metabolic switch to heterotrophy. On the other hand, Katoh (1983) has shown that Marchantia polymorpha L. was not able to grow in the dark, even in the presence of a carbon source (glucose), due to reduced respiration levels. There have also been experiments in which sugar has induced sexual organs, such as in B. argenteum (Liang et al. 2010). However, in our study, no such phenomena were observed. The gathered results suggest that the role of sugars in bryophyte develop- ment can vary significantly among species and that a deeper understanding of these species-specific responses will be crucial for optimizing growing condi- tions. Further research is needed to elucidate the potential role of sugars in the metabolism of selected moss species. Increasing the BAP concentration consistently led to a decrease in the for- mation of new shoots in both upright and prostrate-oriented explants (Fig. 4C). Similar findings were observed in studies on D. lycopodioides (Jadranin et al. 2024) but also in the acrocarpous mosses H. heimii, B. argenteum, A. undula- tum, and P. cuspidatum (Sarla and Chopra 1987; Sabovijevié et al. 2022 and the references therein). In contrast to these results, a large number of newly formed gametophores were observed in P patens when exogenous BAP was applied (Ashton et al. 1979). Secondary protonema was mostly inhibited when BAP was applied, except in the prostrate-oriented explants treated with the lowest concentration (0.03 uM) (Fig. 4D). These findings were previously documented for Garckea phascoides (Hook) C. Muller (Chopra and Sarla 1986), E. pulchellus (Cosié et al. 2025b) and P. sibiricum (Jadranin et al. 2023). Such results sug- gest that BAP is not suitable for the propagation of P krylovii under axenic con- ditions, as the plant can spontaneously produce more shoots without the addi- tion of cytokinins. Besides its effects on the formation of protonemal and shoot buds in bryophytes, cytokinins seem to have a role in the formation of female sex organs (archegonia) at the tip of the gametophores, such was the case in the upright-positioned plantlets of D. lycopoidoides (Jadranin et al. 2024). An earlier study conducted on female clones of Bryum argenteum revealed that exogenous cytokinins increased the production of fertile gametophores, while Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 304 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii inhibited it in the male clone (Bhatla and Chopra 1981). Although BAP was not suitable for multiplication in vitro, its application was valuable for the induction of sex organs in the case of this pleurocarpous moss. Overall, the development of new buds can be altered by exogenously added cytokinins and auxins, since there are complex interactions between endoge- nous hormones and the exogenous portion of PGRs. Therefore, due to the spe- cies-specific responses of bryophytes tested so far of the PGRs, further studies are required, making the determination of optimal concentrations a challenge. However, the results clearly indicate that P krylovii can be propagated vege- tatively and axenically on basal KNOP medium without the addition of PGRs. However, because of the various effects that PGRs can have in bryophytes, it is important to study their potential effects on the formation of some other struc- tures besides the impact they have on morphogenetic parameters. The results achieved in this study clearly introduce new knowledge to the biol- ogy as well as the ex situ/in vitro growth conditions of this rare species. However, the axenic cultures and propagation offer many further possibilities for investi- gations like biological activity and chemical constituents of this moss species as well as other biological features like biofiltration or toxic elements accumulation. Conclusion The rare and threatened moss P krylovii was successfully propagated under axenic and xenic ex situ conditions and optimal conditions for biomass produc- tion were achieved. The study shows that the KNOP medium is most suitable for the propagation of P krylovii, especially when the explants are placed in a prostrate orientation, which gives the highest index of multiplication. This orientation enhances nutrient uptake, resulting in better biomass production. The BCD medium supports growth, but its effectiveness is lower compared to KNOP. The MS/2 medium, on the other hand, is unsuitable as the plants in- vested more in the development of the secondary protonema and developed a lower number of new shoots. The addition of different concentrations of IBA had no effect on the index of multiplication, while the addition of BAP led to a lower formation of new shoots and inhibited the development of protonema. Furthermore, the tested sugars had no positive effect on the multiplication of an axenic culture of P krylovii, and sucrose even reduced the number of newly formed shoots. Leafy gametophores were successfully used for acclimation and xenic ex situ propagation of the species. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding The project is supported by the Serbian Ministry of Science, Technological Development and Innovations, contract no: 451-03-65/2024-03/2001 78 and 451-03-66/2024-03/2001 78. Nature Conservation 58: 289-309 (2025), DOI: 10.3897/natureconservation.58.150920 305 Bojana Z. Jadranin et al.: Ex situ conservation of Podperaea krylovii Author contributions Conceptualization, A.D.S. and M.S.S.; Methodology, M.V.C and M.M.V.; Software, D.P.B.; Validation, A.D.S., D.P.B., B.Z.J and M.S.S.; Formal analysis, B.Z.J., M.V.C and D.PB.; In- vestigation, B.Z.J., M.V.C and D.P.B.; Resources, M.S.I, A.V.T and M.S.S.; Data curation, B.Z.J. and D.P.B.; Writing—original draft, B.Z.J., M.V.C and D.P.B.; Writing-Review and Ed- iting, B.Z.J., M.V.C D.PB. and M.S.S.; Visualization, B.Z.J., M.V.C and D.PB.; Supervision, A.D.S. and M.S.S.; Project administration, A.D.S.; Funding Acquisition, M.S.S. Author ORCIDs Bojana Z. Jadranin © https://orcid.org/0009-0001-4506-699X Marija V. Cosié © https://orcid.org/0000-0002-3506-8705 Djordje P. Bozovié © https://orcid.org/0000-0002-5816-5903 Milorad M. Vujicié © https://orcid.org/0000-0002-21 52-9005 Michael S. Ignatov ® https://orcid.org/0000-0001-6096-6315 Aleksey V. Troitsky © https://orcid.org/0000-0002-0471-2633 Aneta D. 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