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For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant

Ex situ conservation of Bulgarian endemic plant Achillea thracica Velen. was achieved by successful in vitro cultivation of mono-nodal segments on MS-B5 medium supplemented with 1.0 mg/L BA for 20 days and subsequent transferring of regenerated plants on hormone free basal MS-B5 medium for root development and accumulation of leaf biomass. In vitro multiplicated plants were successfully acclimated in a growth chamber with 100% survival. GC–MS analysis of the essential oils resulted in the identification of 30, 10 and 28 compounds in in situ grown, in vitro cultivated and ex vitro adapted plants, respectively, constituting 77.7%, 99.9% and 84.1% of the total oils. The wider variety of compounds was found in the essential oils of in situ and ex vitro adapted plants where santolina alcohol, β-eudesmol, 1,8-cineole, germacrene D, α-cadinol and artemisia alcohol were the principal components comprising 68.7% and 69.3 of the oil, respectively. In vitro cultivated plants consist of mainly 1,8-cineole, germacrene D and artemisia alcohol representing 87% of the oil. Different growth conditions affect the composition of essential oils, suggesting their possible involvement in the process of adaptation and surviving in changing environmental conditions.

The genus Teucrium includes perennial herbs or shrubs widespread all around the world. The wall germander (Teucrium chamaedrys L.) is widely used in traditional and modern medicine for various health disorders and recent research revealed that serpentine populations of this herb have increased antioxidant and respective medical potential compared with calcareous population. The present study aims to elucidate further the role of phenolic compounds for T. chamaedrys growth on serpentine soil. Methanol extracts from two serpentine and two non-serpentine (calcareous and siliceous) Bulgarian populations were obtained and analysed for total quantity of phenolics and flavonoids, and antioxidant activity. The serpentine soils acted as an elicitor of the antioxidant response in T. chamaedrys plants and the increased phenolics level correlated with enhanced antioxidant activity. The calcareous population had lower values but still higher than the siliceous population which had the lowest antioxidant values, although the flavonoid quantity that was equal to the serpentine populations. A gas chromatography-mass spectrometry (GC-MS) assay for identifying phenolic acids pointed to caffeic acid as a dominant compound, with additional cinnamic and benzoic acid derivatives with putative roles as antioxidants, in cell wall lignification for heavy metals chelation, and for interspecies interactions.

Hairy root (HR) culture is considered as "green factory" for mass production of bioactive molecules with pharmaceutical relevance. As such, HR culture has an immense potential as a valuable platform to elucidate biosynthetic pathways and physiological processes, generate recombinant therapeutic proteins, assist molecular breeding, and enhance phytoremediation efforts. However, some plant species appear recalcitrant to the classical Agrobacterium rhizogenes transformation techniques. Sonication-assisted Agrobacterium-mediated transformation (SAArT) is a highly effective method to deliver bacteria to target plant tissues that includes exposure of the explants to short periods of ultrasound in the presence of the bacteria.Nuclear magnetic resonance (NMR)-based metabolomics is one of the most powerful and suitable platforms for identifying and obtaining structural information on a wide range of compounds with a high analytical precision. In terms of plant science, NMR metabolomics is used to determine the phytochemical variations of medicinal plants or commercial cultivars in certain environments and conditions, including biotic stress and plant biotic interaction, structural determination of natural products, quality control of herbal drugs or dietary supplements, and comparison of metabolite differences between plants and their respective in vitro cultures.In this chapter, we attempt to summarize our knowledge and expertise in induction of hairy roots from rare and recalcitrant plant species by SAArT technique and further methodology for extraction of secondary metabolites of moderate to high polarity and their identification by using NMR-based metabolomics.

Hairy root (HR) culture is considered as "green factory" for mass production of bioactive molecules with pharmaceutical relevance. As such, HR culture has an immense potential as a valuable platform to elucidate biosynthetic pathways and physiological processes, generate recombinant therapeutic proteins, assist molecular breeding, and enhance phytoremediation efforts. However, some plant species appear recalcitrant to the classical Agrobacterium rhizogenes transformation techniques. Sonication-assisted Agrobacterium-mediated transformation (SAArT) is a highly effective method to deliver bacteria to target plant tissues that includes exposure of the explants to short periods of ultrasound in the presence of the bacteria.Nuclear magnetic resonance (NMR)-based metabolomics is one of the most powerful and suitable platforms for identifying and obtaining structural information on a wide range of compounds with a high analytical precision. In terms of plant science, NMR metabolomics is used to determine the phytochemical variations of medicinal plants or commercial cultivars in certain environments and conditions, including biotic stress and plant biotic interaction, structural determination of natural products, quality control of herbal drugs or dietary supplements, and comparison of metabolite differences between plants and their respective in vitro cultures.In this chapter, we attempt to summarize our knowledge and expertise in induction of hairy roots from rare and recalcitrant plant species by SAArT technique and further methodology for extraction of secondary metabolites of moderate to high polarity and their identification by using NMR-based metabolomics.

Lamium album L. is a perennial herb widely used in folk medicine. It possesses a wide spectrum of therapeutic activities (anti-inflammatory, astringent, antiseptic, antibiotic, antispasmodic, antioxidant and anti-proliferative). Preservation of medicinal plant could be done by in vitro propagation to avoid depletion from their natural habitat. It is important to know whether extracts from L. album plants grown in vitro possess similar properties as extracts from plants grown in vivo. For these reasons, it is important to examine changes in the composition of secondary metabolites during in vitro cultivation of the plant and how they affect the biological activity. We used A549 human cancer cell line and normal kidney epithelial cells MDCKII (Madin–Darby canine kidney cells II) as controls in assessing the anti-cancer effect of plant extracts. To elucidate changes in some key functional characteristics, adhesion test, MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER), immunofluorescence staining and trypan blue exclusion test were performed. Methanol and chloroform extracts of in vivo and in vitro propagated plants affected differently cancerous and non-cancerous cells. The most pronounced differences were observed in the morphological analysis and in the cell adhesive properties. We also detected suppressed epithelial transmembrane electrical resistance of MDCK II cells, by treatment with plant extracts, compared to non-treated MDCK II cells. A549 cells did not polarize under the same conditions. Altered organization of actin filaments in both cell types were noticed suggesting that extracts from L. album L. change TER and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II cells.

Objective: Human Herpes Virus (HHV) type 1 and 2 are cause of hidden pandemics in global scale, as well as sever clinical symptoms associated with active replication in the human host. As until now there are 11 license anti-herpes drugs. Most of them are based on acyclovir and his derivative. Their frequent usage leads to the selection of drug resistance strains and patients offen experience unwanted side effects. Natural products (for ins. plant extracts) are tolerated better by living organisms and their complex composition prevent appearance of resistant virions. The aim of our work is to study the effect of Stachys Thracica Dav extracts against Human Herpes Virus type 1, strain F and Human Herpes Virus type 2, strain BA.Materials and methods: The extracts are obtained from in vivo, in vitro and ex vitro cultivated plants, using methanol extraction. All tests are done in in vitro experimental settings. We use MDBK cell line, and also laboratory strain F of HHV – 1. The following methods were applied: MTT assay to determine cell survival, direct contact assay to test virucidal activity and modified MTT assay to determine effect against virus replication in cell culture. Results: Obtained data shows that two of the extracts – those from in vitro and ex vitro cultivated plants are with close MNC (maximal nontoxic concentration) (2mg/ml), and the third one is more toxic (MNC is 1mg/ml). The tested extracts do not influence the replication of HHV – 1 and HHV-2. The results about virucidal activity show that the extracts strongly inhibited extracellular virions of HHV-1, strain F (extracts from in vitro cultivated plants reach ~ 100% at 240 minutes of the contact), but the effect of the extracts on extracellular virons of HHV-2, strain BA is more slight.Conclusions: Tested extracts do not have effect against virus replication in cell culture but show strong virucidal activity against HHV-1 and slight activity against HHV-2.

Antioxidative activity of two in vitro cultivated Hypericum species - H. rumeliacum Boiss. and H. tetrapterum Fr. - was estimated after cryopreservation. Both species were successfully regenerated after a cryopreservation procedure performed by the vitrification method. H. tetrapterum did not manifest any significant oxidative stress-induced changes caused by low-temperature treatment. Conversely, a decrease in green pigments' content of H. rumeliacum was measured, particularly pronounced in chlorophyll b, which was accompanied by an increase of carotenoids in the regenerated plants. A strong increase of malone dialdehyde and H2O2 levels in H. rumeliacum tissues was detected. Superoxide dismutase activity was enhanced by 170%, as well as the catalase activity, which was 220% above the control. The same trend was observed in H. tetrapterum, although less pronounced - 143% increase of superoxide dismutase and 112% of catalase. Cryopreservation did not influence the phenol content in the examined plants, but it led to an increase of flavonoid content, especially in H. tetrapterum, by 237%. Total antioxidant activity in regenerated H. tetrapterum varied around the control level, but it was increased in H. rumeliacum. The free proline content in H. tetrapterum remained almost unaffected after freezing, as opposed to H. rumeliacum, where a strong increase of proline content (208% above the control) occurred. An electrolyte leakage from the cells of H. rumeliacum regenerated after cryopreservation was also registered, albeit not significant.

Lamium album, commonly known as white dead nettle or non-stinging nettle is a flowering herbaceous plant, native throughout Europe, Western Asia and North Africa. From ancient times this plant has been endowed with revival, curative and culinary virtues. In the past, in the traditional and folk medicine white dead nettle has been used mainly for its anti-inflammatory, astringent and anti-septic activity. Nowadays significant amount of knowledge on the efficacy of extracts and raw material of L. album is accumulated and a number of health-related beneficial activities have been scientifically proven. In vitro analyses conducted in various model systems have demonstrated antiviral, antimicrobial, antioxidant, anticancer, cytoprotective, wound healing and other important pharmacological effects. The present review summarizes the recent information on the phytochemical features of this pharmacologically important species. The findings on the chemical composition, biological activities and the pharmacological properties underlying the revival secret of white dead nettle are described and discussed in the view of potential applications for treatment of human diseases. Trends for further research are outlined.

To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

The effect of different concentrations (0.1-1.0 mg/L) of cytokinin BA (6-benzylaminopurine) and auxin IBA (indole-3-butyric acid) on the in vitro propagation and on the amount of secondary metabolites accumulation as well as their antioxidant capacity in the medicinal plant Lamium album was examined. A slight increase in shoot numbers was observed on Murashige and Skoog’s (MS) medium supplemented with 0.8 mg/L BA when compared to control plants that were propagated on regular MS medium. All concentrations of BA suppressed root formation and stimulated callogenesis. Significant root formation on the MS medium supplemented with different concentrations of IBA was observed, especially at 0.7 mg/L IBA. Shoots cultivated on MS medium supplemented with BA from 0.6 up to 0.8 mg/L and IBA in concentration of 0.9 mg/L showed enhanced content of total phenols and flavonoids. The highest values of antioxidant activity were achieved on MS medium with 0.8 mg/L BA and 0.9 mg/L IBA. Compared to shoots and in vivo growing plants, significant stimulation of total phenolic content and antioxidant activity in forming callus and roots occurred on MS medium supplemented with 0.2, 0.5, 0.8 mg/L BA and 0.2, 0.5, 0.7 mg/L IBA. The amount of flavonoids in developed callus was twofold lower than the one observed in shoots. As far as we know, this is the first report to indicate stimulation activity of BA and IBA on the production and accumulation of phenolic compounds with high antioxidant capacity, especially in roots and callus, in in vitro propagated Lamium album.

Detached tobacco leaves were infiltrated with an AT toxin preparation from the foliar pathogen Alternaria alternata tobacco pathotype. The AT toxin preparation caused formation of necrotic lesions within 5 days post-infiltration in a concentration-dependent manner. Cell death was accompanied by increased levels of the stress metabolites hydrogen peroxide, malondialdehyde, free proline and by enhanced total protease activity. Lesion development and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with caspase-specific peptide inhibitors (irreversible caspase-1 inhibitor acyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and the broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB)), the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethylketone and the polyamine spermine. Extensive accumulation of reactive oxygen species (ROS), as determined by staining with 3-3'-diaminobenzidine and 2',7'-dichlorofluorescein diacetate, was found in the AT toxin-challenged lesions. The data show that AT toxin-induced cell death in tobacco is a type of programmed cell death in which caspase-like proteases and ROS signalling play a prominent role.

The present study reports the chemical composition of headspace volatiles (HS) and acetone extracts of the endemic Bulgarian species Achillea thracica Velen. from its natural habitat (N), in vitro propagated (IN) and ex vitro established (EX) plants. Additionally, acetone extracts were tested by a disk diffusion method for antibacterial activity. Irregular monoterpenes were the most abundant HS volatile constituents, while O,O-dimethyl quercetin was the most abundant flavonoid in the acetone extracts. The secondary metabolites of A. thracica grown in its natural habitat (N), propagated in vitro (IN) and ex vitro established (EX) showed that the qualitative composition is mutually similar, but there are differences in the quantitative composition. Considering antibacterial activity, IN and EX samples showed moderate activity against Pseudomonas aeruginosa and Escherichia coli.

†Background and Aims Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. †Methods Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. †Key Results MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. †Conclusions In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways.

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)-induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene- and NO-interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP-induced cell death. By employing a carbon dioxide laser-based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)-based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP-induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP-induced cell death.

Plant-pathogen interactions are associated with plant defense mechanism known as hypersensitive response (HR), which is a form of programmed cell death (PCD). In the present work we have tested the potency of chemicals, proven as PCD inhibitors in other systems, to prevent the spread of cell death in detached tobacco leaves challenged with ß- xylanase from Aspergillus awamori K1. Xylanase induced cell death that was accompanied by electrolyte leakage and increased levels of the stress metabolites hydrogen peroxide and malondialdehyde. Lesion development, ion leakage and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB), cysteine protease inhibitor iodoacetamide (IA), serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), the antioxidant l-galactone-¿-lactone (L-gal) and the ethylene blocker (S)-trans-2-Amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride (AVG). Massive ROS accumulation, as determined by staining with 3-3¿-diaminobenzidine and 2¿,7¿-dichlorofluorescein diacetate, occurred in xylanase-infiltrated lesions and was substantially reduced by the inhibitors. To the best of our knowledge, this is the first report showing that ß- xylanase produced by Aspergillus awamori K1 induces cell death response in tobacco and proteolysis, ROS and ethylene are involved in the mediation of the signaling.