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Cistus creticus subsp. creticus is a shrubby Mediterranean plant used since ancient times in folk medicine for the treatment of different diseases. C. creticus extracts and resin contain different types of secondary metabolites, such as terpenoids (predominantly labdane type diterpenoids), and phenylpropanoids. Growth conditions seem to influence the content of labdane-type diterpenes and flavan-3-ols in leaves of C. creticus subsp. creticus. Histochemical staining of leaves’ trichomes and comprehensive phytochemical characterization of resin, leaves and their exudates, indicated that long-stalked capitate trichomes of C. creticus subsp. creticus, grown both in vitro (IV) and in greenhouse (GH), are capable of producing bioactive oleoresin-related terpenoids and phenylpropanoids compounds. Bioactivity-guided approach was implemented in search for the major antibacterial compound in C. creticus resin against two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus cereus and Micrococcus flavus). Bioautographic assay on TLC plates with separated components of Cistus resin extract, revealed a pronounced zone of microbial growth inhibition, corresponded to a highly active compound with Rf values of 0.45, structurally characterized and identified as ent-3β-acetoxy-13-epi-manoyl oxide. This finding opens the route for focusing on isolation and functional characterization of genes involved in the biosynthesis of ent-3β-acetoxy-13-epi-manoyl oxide and its precursor ent-3β-hydroxy-13-epi-manoyl oxide, with the aim to establish sustainable in vitro biotechnological protocols for its large-scale production in homologous and heterologous plant and microbial hosts.

Simple Summary Severe environmental changes, such as drought, can delay growth, the development of plants, and induce injury to their tissues. However, a group of land plant species, called resurrection or desiccation-tolerant plants, is able to lose 95% of their cellular water and still remain viable for long periods, resuming full metabolic activity upon rehydration. Recovery from near-complete water loss is complex and requires the coordination of physical and chemical processes in the resurrection plants. Under stress conditions plants also synthesize and release a wide variety of volatile organic compounds with diverse biological and ecological functions. The rehydration process in resurrection rustyback fern (Asplenium ceterach) resulted in complete plant recovery within 72 h, accompanied by high emission of volatiles, mainly belonging to the group of fatty acid derivatives. These findings could have significant implications from biotechnological and ecological perspectives since the rustyback fern has been recently recognized as a valuable source of bioactive compounds. Abstract When exposed to stressful conditions, plants produce numerous volatile organic compounds (VOCs) that have different biological and environmental functions. VOCs emitted during the rehydration process by the fronds of desiccation tolerant fern Asplenium ceterach L. were investigated. Headspace GC–MS analysis revealed that the volatiles profile of rustyback fern is mainly composed of fatty acid derivatives: isomeric heptadienals (over 25%) and decadienals (over 20%), other linear aldehydes, alcohols, and related compounds. Aerial parts of the rustyback fern do not contain monoterpene-type, sesquiterpene-type, and diterpene-type hydrocarbons or corresponding terpenoids. Online detection of VOCs using proton-transfer reaction mass spectrometry (PTR–MS) showed a significant increase in emission intensity of dominant volatiles during the first hours of the rehydration process. Twelve hours after re-watering, emission of detected volatiles had returned to the basal levels that corresponded to hydrated plants. During the early phase of rehydration malondialdehyde (MDA) content in fronds, as an indicator of membrane damage, decreased rapidly which implies that lipoxygenase activity is not stimulated during the recovery process of rustyback fern.

The impact of ploidy level on both the regenerative potential under in vitro conditions and the production of major bioactive specialized metabolites, such as iridoids and xanthones, was examined in Centaurium erythraea Rafn. Shoot regeneration frequency was genotype dependent, but not affected by explant ploidy level. In most cases, the regenerated shoots of autotetraploid (4x) genotypes were more robust than diploid (2x) ones. Regeneration efficiency of root explants declined from the apical to the basal root segment. Shoot and root biomass production of two month-old plants was not significantly different between 2x and 4x genotypes. Both 4x and 2x genotypes were characterized by the predominance of secoiridoid glucoside gentiopicrin in shoots and roots, which is followed by swero-side and swertiamarin. Loganic acid, loganin and secologanin were much less abundant. Methylbel-lidifolin was the major xanthone in both shoots and roots. Diploid plants showed higher biosynthetic capacity for the production of secoiridoids and xanthones in both shoots and roots. Results highlight a higher potential of diploid C. erythraea genotypes for biotechnology-based sustainable production of secoiridoids in comparison to tetraploid genotypes.

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

A number of Nepeta species (fam. Lamiaceae) are interesting medicinal crops for arid and semi-arid areas, due to their ability to maintain essential developmental and physiological processes and to rationalize their specialized metabolism under water deficit growth conditions. The present research is, to our knowledge, the first attempt to investigate the molecular background of the dehydration-induced changes in specialized metabolism of Nepeta species, which will help to understand relations between dehydration stress on one hand and biomass production and yield of nepetalactone (NL) on the other. During the 6 days exposure of Nepeta rtanjensis Diklić & Milojević and Nepeta argolica Bory & Chaub. ssp. argolica plants to PEG-induced dehydration stress under experimental in vitro conditions, decrease in transcript levels of the majority of 10 NL biosynthetic genes, and some of the 5 transcription factors (TFs) were recorded, simultaneously with the initial reduction in NL content. The two model species evidently employ similar strategies in response to severe dehydration stress; however N. rtanjensis is highlighted as the species more efficient in maintaining NL amounts in tissues. The results suggest trichome-specific and co-ordinately regulated NL biosynthesis at the level of gene expression, with trichome enriched MYC2 and YABBY5 TFs being the potential positive regulators. Manipulation of such TFs can be effective for engineering the NL biosynthetic pathway, and for the increased production of cis,trans-NL in N. argolica ssp. argolica and trans,cis-NL in N. rtanjensis.

Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of the established bioinformatics approach by amplifying them in eight different taxa within the genus Centaurium having various ploidy levels (diploids, tetraploids and hexaploids). Nine markers displayed polymorphism and/or transferability among studied taxa. They provided 54 alleles in total, ranging from 2 to 14 alleles per locus. The highest number of alleles was observed in C. erythraea, C. littorale and a hybridogenic taxon C. pannonicum. The developed markers are qualified to be used in genetic population studies on declining natural populations of Centaurium species, thus providing valuable information to evolutionary and conservation biologists. The developed cost-effective methodology provides abundant de novo assembled short contigs and holds great promise to mine numerous additional EST-SSR-containing markers for possible use in genetics population studies of tetraploid taxa within the genus Centaurium. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173024]

Nepeta rtanjensis Diklic & Milojevic (fam. Lamiaceae) is an endemic, critically endangered plant, protected by the law in Serbia. Various biological activities have been ascribed to its major constituents, nepetalactones. In this study we describe for the first time cytotoxic activity of N. rtanjensis essential oil (EO), obtained from field cultivated plants, which was found to be especially rich in trans,cis-nepetalactone. MTT assays indicated that after 72 h of treatment the EO exhibited cytotoxic activity on investigated cancer cell lines: HeLa, K562, A549, LS-174 and MDA-MB-231. Normal cell line (MRC-5) was the least sensitive to the treatment and IC50 value for this cell line was not reached within the tested range of EO concentrations (up to 0.1 μL/mL). Analysis of morphological changes of treated cells confirmed the higher sensitivity of tumor cells than normal cells to the tested EO. Application of N. rtanjensis EO resulted in the appearance of morphological changes in tested cancer cell lines characteristic for apoptotic cell death, and induced perturbations of the cell cycle of HeLa cells. In addition, upregulation of Bax and p53, and downregulation of Bcl-2, and Skp2 genes, involved in apoptotic signalling cascades, confirmed an apoptosis-inducing effect of N. rtanjensis EO on HeLa cells. Presented results highlighted the potential of N. rtanjensis EO in anticancer therapy.

Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression pattern was observed for CeBIS1, imposing itself as the TF that is prominently involved in wound-induced regulation of SGs biosynthesis genes. The high degree of positive correlations between and among the biosynthetic genes and targeted TFs expressions indicate the transcriptional regulation of SGs biosynthesis in response to wounding with a significant role of CeBIS1, which is a known component of the jasmonic acid (JA) signaling pathway.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.

Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression pattern was observed for CeBIS1, imposing itself as the TF that is prominently involved in wound-induced regulation of SGs biosynthesis genes. The high degree of positive correlations between and among the biosynthetic genes and targeted TFs expressions indicate the transcriptional regulation of SGs biosynthesis in response to wounding with a significant role of CeBIS1, which is a known component of the jasmonic acid (JA) signaling pathway.

While bioactive properties of Centaurium erythraea Rafn secoiridoid glucosides (SG) are widely recognized, many aspects related to their biochemistry, metabolism and relationship to the overall plant physiology are not yet understood. Here we present for the first time an insight into the molecular background of organ-specific and genotype-dependent constitutive biosynthesis of secoiridoids in C. erythraea, by comparing chemical profiles and secoiridoid glucosides-related gene expression. Genes encoding enzymes for intermediate steps of secoiridoids biosynthesis up to secologanin have been identified by analysing transcriptomic data from C. erythraea leaves. Results suggest an organ-specific capacity for the production and accumulation of secoiridoid glucosides, and highlight leaves as the main biosynthesis site. They also point out that significant differences in SG content among various C. erythraea genotypes, are, at least partially, determined by different expression patterns of SG-related genes. The biosynthesis of SG in C. erythraea leaves is enhanced upon treatments with methyl jasmonate (MeJA), which causes reprogramming of SG-related gene expression, leading to an increased production of valuable bioactive compounds. The present study unveiled several rate-limiting genes (encoding GES, G8O, 8HGO, IS and 7DLGT) in SG biosynthesis. SLS and CPR are highlighted as important genes/enzymes that might regulate biosynthetic flux through SG pathway. Information gathered within this study will help us gain deeper insight into the SG metabolism and develop strategies for enhanced biosynthesis of specific secoiridoid glucosides in homologous or heterologous systems.

Nepetalactones are accumulated as the main secondary metabolites in the majority of Nepeta species; however, their biosynthetic pathway remains to a great extent unknown, as well as their relationship to the overall physiology of these plants. Nepetalactones most certainly arise through general iridoid biosynthetic pathway leading to nepetalactol, a common precursor of all plant-derived iridoids that is formed in a reaction catalysed by iridoid synthase (IS, EC 1.3.1.99). In this paper, we aimed to localize the sites of nepetalactone biosynthesis and accumulation by comparatively analysing the nepetalactones content and the expression of putative iridoid synthase (NrIS) in leaves of Nepeta rtanjensis Diklic & Milojevic, a trans,cis-nepetalactone and dehydronepetalactone rich species. Another aim was to give an insight into the developmental regulation of nepetalactone biosynthesis in leaves of cultivated N. rtanjensis plants. Knowledge on the spatial-, and developmental- regulation of nepetalactone metabolism could greatly benefit potential growers by enabling them to adequately plan and manage plant cultivation, harvesting, and handling and thus increase yield of bioactive compounds. The expression profile of putative NrIS1 is highly correlated with trans,cis-nepetalactone and dehydronepetalactone content/ratio in leaves of different developmental stages. The results strongly indicate that developmental regulation of nepetalactone biosynthesis in leaves resides largely at the level of gene expression, and suggest an important role of NrIS1. Young leaves exhibited the most intensive nepetalactone biosynthesis and accumulation, but also its conversion to dehydronepetalactone. Thus, the pattern of NL accumulation in leaves can be explained by both the rate of its biosynthesis and metabolic degradation. The gene expression patterns of NrIS1 and NrIS2 transcripts in trichomes and abraded leaves revealed trichomes as the main site of their activity. UHPLC/qqqMS profiling of nepetalactones in trichomes and abraded leaves, complemented with Raman spectroscopy and dichloromethane dipping experiment, supported glandular trichome-specific localization of nepetalactone biosynthesis and accumulation.

Chenopodium murals L. wild-type roots (WR) and hairy roots (HR) exudates have been previously shown to exert allelopathic activity against test plant species lettuce, wheat and Arabidopsis. To further investigate their allelopathic nature, a comparative morpho-histological characterization and total phenolic content (TPC) evaluation were performed. Furthermore, the phytotoxic activity of WR and HR clone R5 against three weed species and their antioxidant responses were also assessed. Except for the higher degree of branching and root hair incidence in HR clones, both WR and HR showed similar anatomical features, typical for higher plants. No significant difference in total phenolic content between WR and HRs, nor their exudates was found. Root exudates of WR and R5, applied as growth medium (GM) wherein these C. murale roots were cultured, displayed selective phytotoxic activity depending on the target weed species, ranging from suppression of germination to inhibition of seedling growth, that were linked with alterations in antioxidant enzyme activities. R5 greatly inhibited germination and seedling growth of Capsella bursa-pastoris, while WR inhibited those of Vicia angustifolia and Amaranthus retroflexus. Such phytotoxic properties (of WR and R5) qualify them as a promising natural resource in the management of weeds. Differences in allelopathic activity between HR and WR should be searched for in their allelochemical profile and the content of each allelopathic substance. The lack of anatomical abnormalities in hairy roots renders them an efficient tool for functional-genomic studies of C. murale root genes. (C) 2014 SAAB. Published by Elsevier B.V. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}ON173024, ON173015]

Asplenium ceterach L. (syn. Ceterach officinarum Willd.) or rustyback fern is a plant species traditionally used in Mediterranean countries as an expectorant, diuretic, against spleen complaints, kidney stones and hemorrhoids. Phytochemical analysis of gametophytes and sporophytes of A. ceterach was performed, followed by comparative analysis of phenolic composition and the antioxidant properties of the extracts (scavenging capacities against ABTS•+ and DPPH•). Totally 16 phenolic compounds belonging to the classes of phenolic acids (hydroxybenzoic and hydroxycynnamic acids), flavonoids (flavan-3-ols, flavonols), and xanthones were identified using UHPLC/DAD/-HESI-MS/MS analysis. Different phenolics' composition of the two phases of the life cycle of this fern was observed which significantly deter- mined their radical scavenging activities. In sporophytes of A. ceterach considerably high amounts of chlorogenic acid were found (~21 µg 100 mg-1 fresh weight), while xanthones were the most abundant in gametophytes (mangiferin glucoside, reaching 2.54 µg 100 mg-1 fresh weight), recommending this fern species as a valuable source of bioactive compounds.

Nepeta essential oil (Neo; catnip) and its major component, nepetalactone, have long been known to repel insects including mosquitoes. However, the neural mechanisms through which these repellents are detected by mosquitoes, including the yellow fever mosquito Aedes aegypti (L.), an important vector of Zika virus, were poorly understood. Here we show that Neo volatiles activate olfactory receptor neurons within the basiconic sensilla on the maxillary palps of female Ae. aegypti. A gustatory receptor neuron sensitive to the feeding deterrent quinine and housed within sensilla on the labella of females was activated by both Neo and nepetalactone. Activity of a second gustatory receptor neuron sensitive to the feeding stimulant sucrose was suppressed by both repellents. Our results provide neural pathways for the reported spatial repellency and feeding deterrence of these repell ents. A better understanding of the neural input through which female mosquitoes make decisions to feed will facilitate design of new repellents and management strategies involving their use. The final publication is now available at: [https://doi.org/10.1093/jme/tjx059]

Secoiridoid glycosides are naturally occurring phytochemicals of great importance for the food and pharmaceutical industry because of their various biological activities. Certain Gentiana and Centaurium species., which are recognized as the most important sources of these compounds, have become critically endangered due to overexploitation. In this study we describe a laboratory-scale approach for further implementation in large-scale production of secoiridoid glycosides, using a hairy root culture system of Centaurium maritimum L. Fritch, an underutilized and phytochemically unexplored species. Hairy roots were induced by Agrobacterium rhizogenes strain A40M70GUS and grown in Erlenmeyer flasks, as well as in RITA temporary immersion bioreactors (TIBs). About 2-4 times higher biomass production rate and up to 8 times higher total secoiridoid glycosides production rate were achieved in RITA bioreactors. Among the selected hairy root lines, line HR3 cultured in RITA (R) TIBs proved to be the most efficient considering both biomass and secoiridoid glycosides production rate. (C) 2013 Elsevier Ltd. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia [173024, 41011]

The present study was aimed to investigate the elicitation possibility of the main secondary metabolites production in Nepeta cataria L. and N. pannonica L. plants, by exposing them to synthetic compounds from the group of tetraoxanes and tiophenes. The effect of DO63 (1,2,4,5-tetraoxane) and DOVF15 (2,5-diphenyl-tiophene) on the production of cis,trans-nepetalactone (NL) and rosmarinic acid (RA) in two Nepeta species, was investigated in shoots grown on culture medium with addition of synthetic compounds in concentrations ranging from 0.1 to 2 mg L-1. The content of targeted metabolites in tested in vitro shoots depended on the type and the concentration of applied synthetic compounds. Application of DO63, primarily in concentration of 0.1 mg L-1 to 1 mg L-1, affected only NL production in both Nepeta species resulting in increased NL content in treated shoots, while production of RA was not influenced. Addition of DOVF15 caused decrease of RA content in N. pannonica shoots and increase in N. cataria shoots, whereas NL production was not affected. The presented results highlight the possibility of DO63 and DOVF15 application for the elicitation of the main secondary metabolites production in species from the genus Nepeta. [Projekat Ministarstva nauke Republike Srbije, br. 172008 i br. 173024]

Cistus creticus subsp. creticus is a native plant of the Mediterranean region and it has been used since ancient times for its medicinal properties. The leaves secrete labdane type diterpenes that exhibit considerable antibacterial and cytotoxic activities. However there are no data about implementation of in vitro culture of C. creticus in order to obtain applicable production of labdane diterpenes. We successfully established in vitro culture of shoots and roots on solid or liquid MS (Murashige and Skoog) medium without addition of plant growth regulators. GC/FID and GC/MS detections were used to carry out characterization of ethanol extracts of shoots and roots from in vitro culture. Labdane diterpenes were the most abundant compounds in shoot extract, but absent from the root extract. Total phenolic and flavonoid contents of C. creticus shoot and root ethanol extracts were also determined. In order to evaluate possible cytotoxic activity of extracts, sulforhodamine B (SRB) assay was performed on five human cancer cell lines. Only shoot extract had cytotoxic activity on HeLa (cervix), MDA-MB-453 (breast) and FemX (melanoma) cancer cells with IC50 reaching 80.83 μg/ml (HeLa), 76.18 μg/ml (MDA-MB-453), and 87.52 μg/ml (FemX) respectively. This is the first report of cytotoxic effect of labdane diterpenes from Cistus on HeLa cells.

The European black poplar (Populus nigra L.) is a tree species that once had the central role in the development of riparian ecosystems in a great part of Europe and Asia. During centuries, it is harassed by the riverside's urbanization, drainage of wetlands, and controlled river management, and compromised by intercrossing with other poplar species and cultivated hybrids. With the aim to perceive its genetic diversity and integrity along the Danube River, 12 natural P. nigra forests from corresponding protected areas were assessed using eight highly polymorphic microsatellite loci and win3 genetic marker. In the sample set, among individuals morphologically recognized as P. nigra, 2.28\% were identified as P. x euramericana hybrids and those were excluded from further analyses. High number of alleles and high heterozigosity have been observed in all populations. Only 2.95 \% of total genetic variance was recorded among populations, while the pattern of genetic differentiation corresponds to the isolation by geographic distance. It was found that one population had experienced a recent reduction in population size. We infer that genetic integrity and variation of European black poplar is not compromised within the studied areas which are, therefore, able to provide high-quality genetic material of nonhybrid P. nigra for maintaining and promoting metapopulations along the Danube River. South East Europe Transnational Cooperation Programme ``DANUBEPARKS STEP 2.0{''}-{''}Anchoring the Danube River Network of Protected Areas as Platform for Preservation of Danube Natural Heritage{''}; Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173024, 173012]

Most of the species from the genus Centaurium Hill readily hybridize between themselves in natural habitats. Artificial interspecific hybrid plants were obtained under in vitro conditions using reciprocal crosses between 4 species: Centaurium erythraea, C. littorale, C. maritimum, and C. pulchellum. Among 76 crossing experiments performed, 3 types of crosses produced seeds and gave 20 interspecific hybrid plants. Nuclear DNA quantification, fingerprinting of molecular markers, and determination of variation in main secondary metabolite content were employed to confirm their hybrid nature. All 3 approaches were capable of detecting hybrid plants. RAPD molecular markers proved to be the most reliable, always placing the hybrids between their parents in dendrograms, while secoiridoid glycoside content also showed intermediate profiles in the hybrid plants in relation to the parents. Hence, each of the techniques employed can be recommended for elucidating the mechanisms of interspecific hybridization in natural populations of Centaurium species in further studies. This could help to comprehend their breeding system and recognize interspecific hybridization as one of the crucial steps in polyploid formation and speciation in this genus. Ministry of Education, Science, and Technological Development of Serbia {[}173024]

Random amplified polymorphic DNA (RAPD) analysis is a simple and reliable method used to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of the assay. In this study, we analyzed the effects of different concentrations of primer, magnesium chloride, template DNA and Taq DNA polymerase to develop and standardize a RAPD protocol for Centaurium species. The optimized PCR reaction mixture included: 50 ng of DNA extracted using a CTbased protocol, 2.5 mM MgCl2, 7.5 pmol primer and 2 U of Taq polymerase in a final volume of 25 ?l. Each of the five primers used in experiments (OPB11, OPB15, OPB18, OPF05 and OPH02) generated reproducible and distinguishable fingerprinting patterns of four Centaurium species. The obtained optimized RAPD protocol and the selected primers are useful for our further work in the genetic diversity studies of Centaurium species.

This study provides the results of the developmental biology of the highly rare and endangered moss species Brachia vogesiaca (recorded in less than 30 localities in the Northern Hemisphere, mainly western, central, and southwestern Europe). The aim of the study was to achieve the fully developed gametophyte and to propagate it for the purpose of conservation, reintroduction, and introduction to potential habitats free from xenic contamination. These gametophytes will be used for the study of genetics and genomics of this species. The micropropagation of B. vogesiaca was successfully applied on BCD medium supplemented with 0.1 mu M BA and on BCD supplemented with 0.3 mu M IBA and 0.3 mu M BA for numerous gametophore production. The highest production of secondary protonema was achieved on MS/2 S/2 medium enriched with 0.1 or 0.3 mu M IBA and 0.3 mu M BA. Rather successfully applied micropropagation of this threatened moss species enables better knowledge of its biology and is of great value for its conservation biology and developmental research. Chemical names used: indole-3-butyric acid (IBA), N-6-benzyladenine (BA), Murashige and Skoog medium (MS). Serbian Ministry of Education and Science [173024, 173030]; Serbian-Hungarian Bilateral cooperation entitled Ex situ conservation of rare and endangered bryophytes in Hungary, Serbia and Europe [TET_10-1-2011-0007]; Spanish Ministry of Science and Innov

Rindera umbellata (Waldst. & Kit.) Bunge is a rare, critically endangered and horticulturally appealing plant with unexplored pharmaceutical potential. Its distribution is restricted to sandy habitats, whereas propagation in nature is limited by fungal infections of the seeds. To initiate its ex situ conservation and provide material for metabolomic studies, we have introduced R. umbellata into in vitro culture using immature embryos as primary explants. A 72% of the embryos germinated on growth regulator-free medium. The optimization of growth conditions was based on varying carbohydrates (sucrose, glucose, and fructose) in the medium. In vitro growth and development of R. umbellata plants were significantly affected by both the type and concentration of the applied sugars. For most recorded parameters, including leaf elongation, biomass production, rooting percentage, and the number and length of roots, 0.1 m sucrose was optimal. The highest percentage of explants with developed buds was achieved on 0.06 m sucrose (38.77%) or 0.3 m glucose (27.43%). The plantlets obtained on 0.1 m sucrose were successfully acclimatized to greenhouse and field conditions with survival rates of 71.43% and 42.86%, respectively. To our best knowledge, this is the first publication dedicated to this species.

The high mountain pottioid moss Molendoa hornschuchiana (Hook) Lindb. ex Limpr. is a very rare and critically endangered bryophyte species in Europe in need for ex situ conservation. A 25-year-old herbarium sample was used to revive and propagate this species for further reintroduction and introduction to potential natural habitats. The reviving of “dead” herbarium specimen was achieved by disposing of axenical organisms as well as adjusting condition for developing secondary protonema, bud inductions, and optimization of gametophyte propagation in vitro condition.The influence of exogenously added growth regulators on the morphogenesis of this species was studied. The plants were cultured in the two basic types of media, viz., BCD and half-strength Murashige and Skoog (MS) supplemented with different concentrations (0.01–0.3 μM) of indole-3-butyric acid (IBA) and N6-benzyladenine (BA) under a 16-h photoperiod. The influence of growth regulators on gametophores multiplication in vitro as well as on protonemal diameter was recorded. Well-developed gametophores were obtained on BCD medium, whereas on half-strength MS medium, secondary protonema was produced, both on hormone-free and supplemented substrate exclusively. Based on multiplication index in vitro, maximum development of gametophores was realized on BCD medium supplemented with 0.3 μM IBA and 0.1 μM BA. However, the widest diameter of secondary protonema was obtained on BCD medium enriched with low concentration of both BA (0.01 and 0.03 μM) and constant concentration of IBA (0.03 μM). Chemical names used: indole-3-butyric acid (IBA), N6-benzyladenine (BA), Murashige and Skoog medium (MS).