The Establishment of an Ex Situ Collection of Primula veris in Bulgaria.

Primula veris is a valuable medicinal plant species with declining populations, protected in Bulgaria by the Biodiversity Act. The present study aimed to increase its extremely low seed germination rate, starting with seeds originating from two Bulgarian populations, and to set up an ex situ field collection. The stimulation effect of three factors was tested in in vivo and in vitro experiments: seeds treated with gibberellic acid (in different concentrations and exposure time), light quality (white, infrared, red, and blue or dark), and cold stratification. The combination of factors resulted in 36 treatment variants in vivo and 8 treatment variants in vitro. No germination was observed in control treatment variants. The highest germinating rate (95%) was noticed in vivo under blue monochromatic light after seed soaking into 0.2% GA3 for 10 h; however, the best results (55% of well-developed seedlings) were observed with a combination of blue light and 0.3% GA3 for 5 h. Seedlings were successfully strengthened in vermiculite in a phytotron, potted in soil and grown in a greenhouse, and then 75 plants were transferred to the field plot, where most of them bloomed at the first vegetation season. These results are intended to serve as a basis for establishing a pilot agriculture of the species.

Evaluation of Glaucine Content in Bulgarian Black Sea Coast Localities of Glauciumflavum Cranz. (Papaveraceae).

The content of the alkaloid glaucine was evaluated in Glaucium flavum plants from seven localities along the Bulgarian Black sea coast during two consecutive years, in order to select those with highest glaucine content. Some fluctuations of glaucine content were observed during the two years, and in most of the localities the alkaloid was lower in 2015. Pomorie and Ahtopol maintained high percentages of glaucine in the dry plant material in the two investigated years, being 2.3% for Pomorie in 2014 and for Ahtopol in 2015. The lowest percentages of glaucine were recorded in the plant material from Shkorpilovtsi (0.9% and 0.6%, respectively in 2014 and 2015). Fluctuations in glaucine content were probably due to some abiotic factors as light, temperature, precipitation, soil substrate, salinity, etc.

Bioactive Compounds in Wild, In vitro Obtained, Ex vitro Adapted, and Acclimated Plants of Centaurea davidovii (Asteraceae).

In vitro cultures were initiated from a single seed of Centaurea davidovii. Whole plantlets were regenerated and cultivated for several months on agar-solidified nutrient media differing by their composition: basal MS medium, MS medium supplemented with plant growth regulators, and liquid MS medium. Plantlets were ex vitro adapted and successfully acclimated to open-air conditions; flowering was observed in some individuals in the first summer, and mass flowering during the second summer. The contents of the total flavonoids and the total phenolic compounds were determined spectrophotometrically in the leaves of the in vitro plantlets cultured on different media, and then compared with those in the leaves of the wild plants and in the leaves of the acclimated plants of the field plot. The sesquiterpene lactone 8α-(5'-hydroxyangeloyl)-salonitenolide was determined by HPLC in leaf samples of C. davidovii wild plants, in vitro obtained plantlets and ex vitro acclimated plants in the greenhouse and on the experimental field plot. The composition of the nutrient medium influenced the contents of all studied bioactive substances. The highest concentrations of all tested secondary metabolites were detected in the leaves of the acclimated plants during mass flowering, the content of the lactone reaching 56.2 mg/g DW, which was several times more than in the other leaf samples. The obtained results revealed both the effectiveness of biotechnological methods for propagation and conservation of rare and endangered plant species, and the possibility to use C. davidovii plants ex vitro acclimated to field conditions as a source of secondary metabolites with potential biological activity.

Influence of nutrient medium composition on in vitro growth, polyphenolic content and antioxidant activity of Alchemilla mollis.

Alchemilla mollis (Buser) Rothm. (Rosaceae) is a high-mountain medicinal plant growing in the Balkan Peninsula, with only one population in Bulgaria. Alchemilla plants (Lady's mantle) are commonly used in traditional medicine for treatment of many gynecological diseases. The commercial drugs "Herba Alchemillae" induce a rapid regeneration of skin epithelium and have styptic and anti-inflammatory actions. Because of the high content of phenolic compounds (tannins and flavonoids) and the ecological plasticity of the species, field cultivation or in vitro biomass production of A. mollis are possible alternatives to its collection from nature. Four MS based nutrient media differing in the concentration of the minerals and supplemented with alpha-naphthaleneacetic acid (NAA) and benzylaminopurine (BAP) were tested in order to examine their influence on the shoot multiplication effectiveness and the antioxidant activity of A. mollis, and also the possible relation between these parameters under the conditions of in vitro culture. The cultures grown for two months on these media differed significantly in their multiplication rates (p < 0.001), as well as in their morphological features--height, leaf color and root development. Methanol extracts of in vitro cultivated and ex vitro adapted and acclimated on Vitosha Mt. (1500 m a. s. l.) plants were analyzed for tannin and flavonoid content and for free radical scavenging activity. The contents of flavonoids and tannins in the in vitro cultures of A. mollis cultivated on the four tested media differed significantly (p < 0.05). The highest flavonoid content was found in the shoots cultivated on the control MS medium, as well as in the ex vitro adapted plants. The antioxidant activity of the in vitro cultures correlated positively with the concentrations of the PGRs in the respective media, and the ex vitro adapted plants had the highest antioxidant activity (IC50 13.1 +/- 1.9 microg/mL) commensurable with that of the commercial antioxidant butylated hydroxytoluene (BHT) used as a positive control, with an IC50 of 12.65 microg/mL.

Galanthamine production by Leucojum aestivum cultures in vitro.

The results described in these studies proved that the successful in vitro bioproduction of galanthamine from L. aestivum shoot-clumps required mainly the selection of in vitro clones with a genetically determined high ability to produce the desired alkaloids, although the expression of this ability could be additionally influenced by diverse exterior factors, such as some components of the nutrient medium, or the cultivation conditions of the ambience. Tissue differentiation was also of great importance for the biosynthetic capacity of the cultures. The most suitable inocula for in vitro biosynthesis of galanthamine in liquid medium were the directly regenerated shoot-clumps, ensuring high alkaloid concentrations between 1 and 2 mg/g DW for the selected clones. We observed astonishing clone-specific dynamics of the biosynthetic activity of all of the studied in vitro clones. The dynamics were obviously related to the strong biological clock of the species, persisting even in several-year old cultures. These dynamics did not coincide with those usual for the plants growing in situ and under controlled field conditions. In our opinion, the clone specificity of the biosynthetic dynamics could be due to the disturbance of the plant regulation mechanism under the equal conditions of the ambience in the culture room. The sharp decrease of the alkaloid concentrations were transient, followed by an increase, so that cultures were retaining their biosynthetic capacity. The biosynthesis of the main alkaloids, galanthamine and lycorine, was influenced by diverse stimulants such as substances causing stress (JA), feeding with alkaloid precursors (the amino acids phenylalanine and tyrosine, and CH), and physical treatment (acoustic waves). However, the course of the biosynthetic dynamics during the period of the treatments was always the most important factor for the success of secondary metabolism stimulation. As far as scaling-up of the in vitro biosynthesis of valuable compounds, a stable and predictable yield is required, and additional investigations aimed at the annulment of the effect plant biological clock on alkaloid biosynthesis are needed. The elucidation of the relative influences of the diverse factors modulating alkaloid biosynthesis was of great importance. The high galanthamine concentrations of the selected in vitro clones are a promising basis for future studies.

Influence of plant origin on propagation capacity and alkaloid biosynthesis during long-term in vitro cultivation of Leucojum aestivum L.

The investigation deals with in vitro clonal propagation of L. aestivum L. (summer snowflake), a threatened Amaryllidaceae plant species in Bulgaria used in the pharmaceutical industry as raw material for production of galanthamine-based medicines. Plants of known origin and with different alkaloid profile were taken from the living collection of the Institute of Botany, Sofia. Bulbs were used to initiate in vitro cultures and 24 clones were multiplied. The influence of the clone origin on the propagation coefficient, shoot and bulblet morphology, alkaloid profile and content of galanthamine, lycorine, and four related alkaloids was evaluated. Clones kept stable alkaloid profiles and for most of them, high regeneration rates were noted. Galanthamine content of some clones was commensurable with that of Bulgarian populations of L. aestivum of commercial importance. Five clones: four galanthamine-type and one lycorine-type were selected as promising for further investigation.

CGC-MS of alkaloids in Leucojum aestivum plants and their in vitro cultures.

Underivatised alkaloid mixtures extracted from intact plants and in vitro cultures of Leucojum aestivum (Amaryllidaceae) were investigated by capillary GC-MS. Excellent peak resolution for the alkaloids was exhibited and isomers of galanthamine and N-formylnorgalanthamine were well separated. Fourteen alkaloids of galanthamine, lycorine and crinane types were identified, 11 in the intact plants and eight in the in vitro cultures.