Temporary immersion systems (TISs) are an advanced biotechnological platform for the large-scale cultivation of medicinal plants and the consistent production of high-value secondary metabolites. In this study, we evaluated three immersion regimes with stand-by periods of 4, 8, or 12 h, each paired with a 15-minute immersion period, to optimise shoot growth and colchicine accumulation in Colchicum autumnale L. and Colchicum bivonae Guss. The 4 h stand-by/15 min immersion regime yielded the highest growth index (C. autumnale: 0.75 ± 0.08; C. bivonae: 1.25 ± 0.03) and maximum colchicine content (C. autumnale: 0.19 ± 0.01 mg/g dry biomass; C. bivonae: 0.25 ± 0.02 mg/g dry biomass). Using gas chromatography-mass spectrometry (GC-MS), detailed metabolic profiling of cultures grown under this optimised regime was performed, resulting in the identification of 46 metabolites, including amino acids, organic acids, sugars, sugar alcohols, phenolic, and fatty acids. Volcano plot analysis revealed 11 upregulated and 5 downregulated metabolites in C. autumnale relative to C. bivonae. Significance analysis of metabolomics (SAM) identified 34 metabolites with statistically significant differences between two species. Hierarchical clustering and partial least squares discriminant analysis (PLS-DA) confirmed clear species separation, with Component 1 explaining 68.8% of the total metabolic variance. Glucose-6-phosphate (VIP = 2.01), citric acid (VIP = 1.85), asparagine (VIP = 1.67), and γ-aminobutyric acid (GABA; VIP = 1.52) were the primary biomarkers differentiating the species. These findings confirm that TISs provide an optimised environment for biomass accumulation and stable alkaloid biosynthesis in the Colchicum genus, with C. bivonae emerging as a promising candidate for biotechnological exploitation.

We report the development of a procedure for ultrasound-assisted microscale extraction of metabolites from the flowers of Saint John’s wort (Hypericum perforatum L.), designed for comparative metabolite analysis of plants from genetic resource collections and natural and segregating populations. The procedure involves high-throughput methanol extraction of metabolites from ground-frozen flowers at a selected stage of flower development, which is carried out in a standard 2 mL Eppendorf tube. A total of 18 compounds, including chlorogenic acid, catechins, glycosylated flavonoids, hypericins, and hyperforin, were identified based on LC/DAD/QTOF analysis, of which 16 could be detected in the UV-Vis spectrum. Two alternative versions of the procedure were evaluated: the “single-flower” procedure, including repeated collection and analysis of single flowers from the tested plant, and the “bulk-flower” procedure, employing the collection of a bulk flower sample from the tested plant and analysis of a portion of the ground sample. The results showed excellent technical reproducibility of the “single-flower” procedure when used with the suggested combination of the peak areas for the proto- and stable forms of pseudohypericin and hypericin. Application of the developed “single-flower” procedure for comparison of the plants derived from seed progeny of the apomictic line Hp93 revealed significantly lower metabolite variation among the apomictic progeny plants compared to the variation observed among plants belonging to different genotypes.

The polyploidization of Hippeastrum papilio influences its primary and secondary metabolism including the biosynthesis of bioactive alkaloids. Hippeastrum papilio is an ornamental plant that has advantages in comparison to the currently used plants for the extraction of galanthamine, a natural compound used for the cognitive treatment of Alzheimer’s disease. In the present study, an autotetraploid line of H. papilio was induced for the first time, after treatment with 0.05

We studied the mode of pollination in Greek oregano (Origanum vulgare ssp. hirtum) under both controlled and open pollination conditions. When grown indoors without the presence of insects, Greek oregano plants did not develop any seeds, indicating a low level of spontaneous self-pollination. Applying manual self-pollination under the same conditions resulted in only 16 seeds, of which only five were able to germinate. At the same time, a clonally propagated Greek oregano plant of the same genotype produced a rich set of over 300 seeds in open field conditions when the flowers were visited by insects in an area where no other Origanum species were observed. Analysis with SSR markers showed that over 70% of the seeds likely resulted from self-pollination, indicating that insect-mediated pollination is essential for the seed development. We further analyzed the cross-pollination of Greek oregano with Common oregano (O. vulgare ssp. vulgare) in open field conditions where the two subspecies were grown in close proximity. Applying SSR markers, we analyzed 83 plants obtained from seeds of three vegetatively propagated Greek oregano mother plants. Surprisingly, the results showed that all analyzed seedlings resulted from cross-pollination of Greek oregano with Common oregano, indicating that cross-pollination between the two subspecies can completely take over the self-pollination or cross-pollination between the Greek oregano plants. The possible impact of the observed high cross-pollination rate on the genetic origin of seeds of selected Greek oregano lines and varieties, as well as on the genetic diversity and structure of natural populations, is discussed.

In the past two decades, the need for a new concept in agriculture has emerged. The new "Greener revolution" should rely on the implementation of sustainable practices in crop production and the achievement of increased yields under the conditions of reduction of water, fertilizer, and pesticide use. The utilization of plant secondary metabolites is viewed by many authors as a possible alternative to synthetic chemicals. Bioactive botanical compounds can be obtained from plants in the form of extracts, essential oils (EO), or both. There are four main groups of plant secondary metabolites depending on their chemical structure: terpenes, phenolics, nitrogen-, and sulfur-containing compounds. A growing body of publications is devoted to the pesticidal properties of various secondary metabolites obtained from plants. The botanical families Meliaceae, Rutaceae, Asteraceae, Annonaceae, Labiatae, and Canellaceae include the most valuable species that are rich in secondary metabolites. The strong fumigant properties of the EOs from many plant species make them attractive in different Integrated Post-Harvest Pest Management systems. Suitable carriers for EOs delivery can be designed using nanoencapsulation. On a worldwide scale, the main botanical insecticides that are commercially available at present are Pyrethrum, Azadirachtin from Neem, and EOs from various plant species. Among the botanicals with considerable antimicrobial activity, there are some successfully authorized and developed commercial phenolics, terpenes, and alkaloids. Among the proven active substances are cinnamaldehyde, l-glutamic acid and gamma-aminobutyric acid, Jojoba oil, essential oils, and others.

In vitro cultures from Narcissus pallidulus and Narcissus cv. Hawera were initiated on MS medium supplemented with different concentrations of BAP and NAA. High multiplication rates were obtained on MS medium supplemented with 6 mg/L BAP and 0.1 mg/L NAA. Callus was induced on MS medium supplemented with 6 mg/L BAP and 2 mg/L NAA. Cultivation of the in vitro cultures on MS medium supplemented with 1 mg/L BAP and 0.1 mg/L NAA or with 1 g/L charcoal resulted in bulb growth. Well-developed bulbs with roots were successfully acclimatized. GC-MS analyses of the alkaloid patterns revealed that Narcissus cv. Hawera biosynthesizes Sceletium-type and Amaryllidaceae alkaloids, while Narcissus pallidulus accumulates only Sceletium-type compounds. The dominant compound in their alkaloid mixtures is mesembrenone which possesses potent antidepressant and antianxiety activities. The easy multiplication of these plants provides excellent possibilities for further studies on mesembrenone biosynthesis and development of technologies for its production, both in vitro and in vivo. (C) 2020 SAAB. Published by Elsevier B.V. All rights reserved.

Rationale Narcissus cv. Hawera has been found to biosynthesize some Sceletium-type alkaloids with antidepressant and anxiolytic activities. This ornamental plant has been poorly studied as a source of bioactive alkaloids including some contraversive reports on in vitro and intact plants. In this study, a detailed GC-MS characterization of its alkaloid fractions is presented. Methods GC-MS was used for the identification of compounds in the alkaloid fractions. Both underivatized and silylated samples were analyzed simultaneously. Elevated plus maze and tail suspension tests were used to assay the anxiolytic and antidepressant activities. Ellman's and MTT-dye reduction assays were used to evaluate the acetylcholinesterase (AChE) inhibitory and cytotoxicity activities, respectively. Results Of the 29 alkaloids, 13 of Sceletium-type were detected. Two new alkaloids were identified as 2-oxo-mesembrine and 2-oxo-epi-mesembrenol. Lycorine was found as a major compound (43.5%) in the crude silylated methanol extract. After the elimination of lycorine by pre-crystallization, the major alkaloids were 40.8% 6-epi-mesembranol, 16.2% 6-epi-mesembrenol, and 13.8% sanguinine. This fraction showed anxiolytic and antidepressant-like activities as well as potent AChE inhibitory and antineoplastic activities. Conclusions Silylation of the alkaloid fractions from Narcissus cv. Hawera provides better separation, structural information, and improved sensitivity for compounds with two and more hydroxyl groups. The lycorine-free alkaloid fraction shows a great potential for further pharmacological studies.

Plant tissue cultures have been widely used in both fundamental and applied types of research on various biological species, and the scientific interest to transfer that technology in industrial scale has been rapidly growing. The use of in vitro technology for commercial propagation of different plant species and the production of bioactive compounds from them has become profitable industry worldwide. In the past decades, the progress in plant tissue culture technology was directed towards the introduction of the liquid medium for cultivation under submerged conditions in different bioreactor types, and automation of the entire process. Some applications of modified bioreactor systems and their importance for the advancement of plant biotechnology in the fields of agriculture, medicine, and pharmacy are discussed in this review.

This paper reviews the recent development of the scientific, legislative, economic and environmental aspects of plant organic farming. The impact of organic farming on biodiversity and soil fertility is discussed in comparison with conventional systems. A significant barrier for wide application and future development of organic farming is the existing diversity of national and international policy instruments in this sector. Special attention is paid to up-to-date research techniques that could help solve a number of the problems typically faced in plant organic farming. It is argued that organic farming is still not productive enough to be considered fully sustainable. This underlines the necessity of strong support for more effective implementation of scientific research innovations and improvement of the networking between all stakeholders – organic producers, scientists and corresponding policy makers at the national and international level.

Gas chromatography-mass spectrometry is one of the base analytical platforms used in plant metabolite profiling. The remarkable recent methodological and technological developments in GC-MS profiling expand the possibilities for its application in different fields of plant science including plant biotechnology. The methods of extraction, fractionation, derivatization, and metabolite identification, associated with GC-MS metabolite profiling, along with examples demonstrating the power and applicability of GC-MS in plant in vitro studies, have been presented in this chapter.

Leucojum aestivum (summer snowflake) is a perennial, wild plant species of medical importance. L. aestivum is in danger of extinction and has been thus put under a regulated regime for conservation. However its natural fields in Bulgaria continue to deteriorate, one of the reasons being contamination of rivers with industrial waste. The plant has been long known not to suffer diseases. In the period 2006-2011, however, diseased bulbs with symptoms of bacteriosis were observed. This paper presents the third of a series of investigations of L. aestivum in Bulgaria with symptoms of bacterial rot.After the pathogenicity tests and on the basis of physiological characteristics, metabolic fingerprints, and PCR with specific primers, bacterial strains causal agents of bacterial rot of L. aestivum were identified as Burkholderia gladioli and Pseudomonas marginalis. Additional PCR using the RAPD technique differentiated Burkholderia gladioli pv. alliicola. It could be speculated that contamination of rivers may have been the reason for the spread of pathogens which normally infect other bulbous plants and can survive in soil and water, thus contributing to deterioration of the natural L. aestivum fields.

Leucojum aestivum, an industrial source of the acetylcholinesterase inhibitor galanthamine, shows a great chemodiversity in its alkaloid synthesis. Samples from various geographically distinct populations from Bulgaria and the Balearic Islands were studied by GC–MS. The alkaloid pattern of the plants of L. aestivum (subsp. pulchellum) from the Balearic Islands were dominated by crinine type compounds. Populations of homolycorine chemotype were distributed along the Danube river in the north part of Bulgaria, which is separated from the south part by the Balkan mountains. Populations with high accumulation of lycorine were found in East Bulgaria near the sea coast, while the south populations were dominated by galanthamine type synthesis. The average of the galanthamine content was found to vary from 0.003 to 0.08% (referred to dry weight) in the north, and up to 0.42% in the southern Bulgarian populations. Some individuals showed up to 0.65% galanthamine. The galanthamine content of the plants from the Balearic island was 0.1% of DW. The galanthamine percentage in the total alkaloid mixture ranged from 0.2 to 95% of the total alkaloids. Our study demonstrated that the geographic isolation of the populations of L. aestivum has led to divergation in the alkaloid biosynthesis and consequently to the occurrence of different chemotypes. This chemodiversity in both alkaloid patterns and galanthamine content provides an opportunity for further selection work toward a galanthamine-rich crop, on the one hand, and makes the species an excellent biological system for molecular studies leading to further improvement of the galanthamine production, which is a valuable alkaloid used in medicine for the treatment of Alzheimer's disease.

Summer snowflake /Leucojum aestivum L./ is a medical bulbous plant which belongs to family Amarillydaceae. It is wild-spread as natural populations in Bulgaria and a valuable source of galantamine. A few virus and fungal diseases have been described on plants of family Amaryllidaceae but only fungal pathogens on summer snowflake. This paper is the first report of Serratia plymuthica and Steno trophomonas maltophilia as causal agents of bulb decay of summer snowflake. To our knowledge, this is also the second report of phytopathogenic isolates of the identified two species.The bacteria were isolated from plants in the region of Tutrakan (Bulgaria) close to the Danube River where pathogenic changes in plant tissues of bulbs of summer snowflake were detected in 2006. Pathogenicity to the natural plant host was confirmed by artificial inoculation. Identification was carried out by biochemical properties and sequence analysis. New hosts upon artificial inoculation of S. maltophilia and S. plymuthica are onion, hyacinth, tulip, narcissus, and crocus.The established bacterial species are known inhabitants of the soil and rhizosphere, contaminants of food and water, human infectious agents, and represent a case, similar to Serratia marcescens, when a species can comprise saprophytic, plant, and human strains simultaneously. Biotechnol. & Biotechnol. Eq. 2012, 26(6), 3338-3344

Leucojum aestivum L. is a threatened medicinal plant used as a source of galanthamine in Bulgaria. The AChE inhibitor galanthamine is applied for treatment of Alzeimer's disease and other neurological disorders. The in vitro storage of genotype accessions from different natural populations with proven alkaloid profiles is of fundamental importance for the protection and rapid propagation of this threatened medicinal plant for both industry and natural resource protection. In this paper we describe a three step protocol for in vitro long-term conservation of L. aestivum- induction, regeneration and storage of explants. A gen-bank in AgroBioInstitute with accessions from 31 Bulgarian populations was created by us with the use of this in vitro protocol.

Leucojum aestivum (summer snowflake) is a plant species used for the extraction of galanthamine, an acetylcholinesterase inhibitor for the treatment of Alzheimer’s disease. Extracts from bulbs collected from 18 Bulgarian populations and from shoot-clumps obtained in vitro from 8 different populations showed variations in their alkaloid composition. Nineteen alkaloids were detected in the studied samples by GC-MS. Typically, the alkaloid fractions of L. aestivum bulbs were dominated by galanthamine type compounds, but lycorine, haemanthamine and homolycorine type alkaloids were also found as dominant compounds in some of the samples. Extracts from the shoot-clumps obtained in vitro were found to contain galanthamine or lycorine as main alkaloids. The galanthamine content ranged from 28 to 2104 μg/g dry weight in the bulbs, and from traces to 454 μg/g dry weight in the shoot-clumps.