Multiplication and Conservation of Threatened Medicinal Plant Arnica montana L. by in vitro Techniques

An efficient and reproducible in vitro protocol for mass production of the threatened medicinal plant Arnica montana L. (Asteraceae) was developed. The effectiveness of various combinations of plant growth regulators on A. montana clonal multiplication was assessed, using seedlings stems as initial explants. Among 12 tested nutrient media, the optimum one (MS supplemented with 1.0 mg/l BAP and 0.1 mg/l IAA) increased the organogenesis frequency up to 95% in the best origin, with mean number of shoots per explant 4.25 for 5 weeks. Sub-cultivations on this medium every 4 weeks led to increase of the propagation rate as in the fifth subculture the average number of shoots per explant reached 12.32+-0.82. Rooting of uniform in vitro shoots was 100% successful on half strength MS medium supplemented with 0.5 mg/l IBA. The ex vitro adapted plants showed 90% survival, and were further acclimatized to two mountain ex situ collections. Plants looked healthy and true-to- type and began to bloom in the second or the third year. In addition, a successful protocol for slow-growth storage of in vitro A. montana cultures was elaborated, after testing 8 media with mannitol or sorbitol. The medium 1/2 MS containing 3% sorbitol and 2% sucrose was chosen as the best one, efficiently retarding the growth of the in vitro plantlets, thus allowing 6-month maintenance without sub-cultivation. The developed in vitro protocols could be of great value for commercial propagation and sustainable conservation of this threatened medicinal plant

Growth and Essential Oils of Salvia officinalis Plants Derived from Conventional or Aeroponic Produced Seedlings

Salvia officinalis L. (Lamiaceae) is cultivated in many countries as a valuable medicinal and aromatic plant with antiseptic and spasmolytic effects due to its essential oils, used as ingredient in many phytopreparations. The present study aimed to enhance the growth of S. officinalis plants and to improve the essential oil quality, applying hydroponic technology. Seeds of cultivated S. officinalis were used as initial material: experimental seeds were germinated on aeroponic vertical system into peat cubes in pots with keramzite and control ones in terrines with ordinary soil. All seedlings were transferred to the greenhouse in pots with sterilized compost and universal soil mixture. The morphometric parameters of both groups, 80 seedlings each, were compared after two months; 36 plants per group were transferred to the field plot, and herbage of 10 randomly chosen plants per group was harvested during the full flowering stage in two consecutive years. Essential oils were extracted on Clevenger apparatus and their composition was analyzed by GC/MS. Conventionally obtained seedlings were significantly higher (P<0.001), while aeroponically derived plants increased their biomass by 18.4%, ramified earlier, and developed much more generative shoots (p<0.05). Plants’ size of the two groups equalized at the second year, essential oils yield being about 1.1% w/v. Some fluctuations in essential oils composition was noticed in the two groups and the two years. To summarize, application of aeroponic system shortened the period from germination to harvest, enhanced plants flowering and reflected on the composition of the essential oils

Phenolic acid profiles of endemic species Verbascum anisophyllum and Verbascum davidoffii

The profiles of methanol extractable and methanol insoluble bound phenolic acids of two species: Verbascum anisophyllum Murb (Balkan endemic) and Verbascum davidoffii Murb. (Bulgarian endemic) were determined. Free radical scavenging activity and total phenolic content of studied extracts and fractions were evaluated by DPPH antioxidant method and Folin–Ciocalteu reagent, respectively. Phenolic acid profiles were analyzed by GC/MS. Sixteen phenolic acids and their derivatives were detected. Ferulic acid was the major individual phenolic acid presented in all extracts and fractions. Hydroxycinnamic, vanillic and p-hydroxybenzoic acids were also abundant in the studied phenolic acid profiles. The presence of gentisic, syringic, isoferulic, dihydroferulic, eudesmic, 3,5-di-tert-butyl-4-hydroxybenzoic acids were reported for the first time to Verbascum species. The greatest variety of phenolic acids was found in the fractions containing methanol insoluble bound hydrolysable phenolic acids. The highest free radical scavenging activity and total phenolic content were established for methanol extractable alkaline hydrolysable fractions. Phenolic acid profiles and free radical scavenging activity of both species were similar although V. davidoffii contained few more components. The present study is the first report on the phenolic acid profiles and free radical scavenging activity of V. anisophyllum and V. davidoffii as well as the first detailed study of the phenolic acid profiles of the Verbacum species

In vitro clonal propagation of Tanacetum cinerariifolium and establishment of an ex situ collection of selected clones

Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae) is a perennial herb endemic to the eastern coast of the Adriatic Sea. The species is widely cultivated in many countries for its bioactive compounds pyrethrins, which are used as natural insecticides. Plants derived from seeds vary greatly in pyrethrin content; therefore, the vegetative propagation of high-quality individuals is very important for the establishment of agricultural pyrethrum crops. The present study deals with rapid in vitro multiplication of pyrethrum, ex vitro adaptation of selected clones and creation of an ex situ collection, as a first step towards introducing the species into agriculture in Bulgaria. Seeds from a private ex situ collection in Bulgaria and from a natural Croatian population were used as initial material for in vitro cultures initiation. Basal MS medium (Murashige and Skoog 1962) or MS supplemented with different concentrations of kinetin and indole-3-butyric acid were used for seed germination and multiplication of one-seed derived clones by consecutive subcultivations. The propagation effectiveness was evaluated as a number of new plants obtained per initial shoot. Considerable losses were noticed due to both endophytic contaminations and necrosis, especially on media supplemented with plant growth regulators. These problems were overcome by medium optimization: adding an antibiotic and modifying the medium to increase the calcium concentration using CaCO3. In the best medium variant (basal MS + 200 mg/L Medaxone + 75 mg/L Ca) no more infected plants were observed, and the percentage of necrotic plants decreased threefold, which resulted in formation of 38.06±10.11 new plants per initial shoot for a period of 7 months. Three hundred and sixty plants were ex vitro adapted in a phytotron (88% surviving rate), then 16 plants from 4 selected clones were transferred to the ex situ collection and bloomed twice from the very first growing season (June and September). The number of the flower heads increased in the second year of field cultivation and an average of 328±138 capitula per plant were counted for the best clone. The first trials to establish a pilot plantation of pyrethrum are promising

Growth and Essential Oils of Salvia officinalis Plants Derived from Conventional or Aeroponic Produced Seedlings

Salvia officinalis L. (Lamiaceae) is cultivated in many countries as a valuable medicinal and aromatic plant with antiseptic and spasmolytic effects due to its essential oils, used as ingredient in many phytopreparations. The present study aimed to enhance the growth of S. officinalis plants and to improve the essential oil quality, applying hydroponic technology. Seeds of cultivated S. officinalis were used as initial material: experimental seeds were germinated on aeroponic vertical system into peat cubes in pots with keramzite and control ones in terrines with ordinary soil. All seedlings were transferred to the greenhouse in pots with sterilized compost and universal soil mixture. The morphometric parameters of both groups, 80 seedlings each, were compared after two months; 36 plants per group were transferred to the field plot, and herbage of 10 randomly chosen plants per group was harvested during the full flowering stage in two consecutive years. Essential oils were extracted on Clevenger apparatus and their composition was analyzed by GC/MS. Conventionally obtained seedlings were significantly higher (P<0.001), while aeroponically derived plants increased their biomass by 18.4%, ramified earlier, and developed much more generative shoots (p<0.05). Plants’ size of the two groups equalized at the second year, essential oils yield being about 1.1% w/v. Some fluctuations in essential oils composition was noticed in the two groups and the two years. To summarize, application of aeroponic system shortened the period from germination to harvest, enhanced plants flowering and reflected on the composition of the essential oils

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 8 alpha-(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

Alternative biotechnological approaches for propagation of Helichrysum italicum (Roth) G. Don (Asteraceae)

Biotechnology uses diverse approaches for plant propagation, among them in vitro micropropagation and hydroponics, based on the plant's capacity for vegetative multiplication. Nowadays, the market demand of Helichrysum italicum is increasing for its valuable essential oils; however, seed quality and crops’ growth are variable. The aim of this study was to test different approaches for rapid multiplication of cultivars. Seeds and stem tips were used as initial material for in vitro cultivation (MS medium), and cuttings - for hydroponic propagation (Cutting board system, GHE) in culture rooms under 16/8 h photoperiod and 23±2°C temperature. In vitro cultures initiation was hampered by high microbial contamination: 55.8% of the seeds survived, and almost all leaf explants dropped out due to fungi or necrosis. Seed germination was stimulated with 0.1% GA3 and increased up to 24.4%, while a fungi-free stem-tips culture was obtained on medium containing antibiotic. Subcultivation was tested on media supplemented with different plant growth regulators, and 6.4 new shoots per explant were obtained on the best medium containing 2 mg/l BAP and 0.2 mg/l NAA, for 8 weeks. Plants cultivated on MS control medium rooted spontaneously and some of them were ex vitro adapted to phytotron and then to а greenhouse. On the hydroponic system, 77.8% of the cuttings rooted; 76.2% of which were transferred to soil mixture, and 81.3% of the potted plants successfully adapted to greenhouse conditions. Vegetative rapid propagation of valuable H. italicum cultivars was proved to be feasible by both in vitro and hydroponic multiplication for a 6-month period

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 8 alpha-(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

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