Embryological features, pollen and seed viability of Arnica montana (Asteraceae) – a threatened endemic species in Europe

The embryological features, mode of reproduction and reproductive capacity (pollen and seed viability) on two naturalized populations of Arnica montana in Bulgaria were studied. The embryological study shows that A. montana is a facultative apomictic species in which sexual reproduction predominates. In this species, it was established that there is a comparatively high viability of the mature pollen and embryos, which enables the successful realization of its reproductive capacity. The results of the study on A. montana reveal that both sexual and asexual vegetative reproduction with rhizomes undoubtedly play more important roles than the apomixis (namely diplospory) for support and preservation of the populations

Dataset : Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species

This is an raw data for the manuscript "Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species "This data includes studies on three endemic species of Alkanet, Alkanna (Alkanna primuliflora, A. stribrnyi and A. graeca) from the family Boraginacea, distributed in Bulgaria. We have presented raw data (photographs, chromatograms and graphs) from embryological, genetic and phytochemical studies. The three Alkanna species are Balkan endemics and have a limited distribution on the Balkan Peninsula. The limited distribution of the species is often associated with problems in the reproductive capacity of the species, which is why the embryological examination was performed. The morphological features that distinguish the three species vary, they are unclear, and sometimes transitional forms are observed between the three species, which is why DNA extraction and Inter-simple sequence repeat (ISSR) amplification product analysis were used. The data included GC of the crude mixtures of alkaloids of Alkanna primuliflora, A. graeca and A. stribrnyi and MS spectra of the identified pyrrolizidine alkaloids (PAs). 1) For the embryological examination (male and female generative sphere, pollen viability and seed germ) a classical paraffin methodology was applied. For the embryological study, we collected flowers and buds at different stages of development from the three species from different populations. The material was processed according to classical methods and documented with photographic material. The main embryological structures and processes in the male and female generative sphere were established after observations using LM “Olympus” CX2. The microphotographs were made using an “Infinity lite” digital camera 1.4 Mpx. The presented photos are unprocessed and are used to describe the male and female and male generative spheres of the three species. The boards in the article are based on these photos. A description is provided to the attached photos in this document. 2) DNA extraction and Inter-simple sequences repeat (ISSR) amplification product analysis of the collected leaves of the three species and applied ISSR patterns of 15 Alkanna individuals belonging to Alkanna primuliflora (1-5), Alkanna graeca (6-10) and Alkanna stribrnyi 11-15) generated by primers (AC) 8YT (left) and (AG) 10C (right), respectively. The DNA marker is GeneRuler 100 bp plus. Based on statistical data processing (Principal Coordinate Analysis (PCoA) and Hierarchical Analysis of Molecular Variance (AMOVA) in GenALEx software) were conducted to show the genetic relationships among the Alkanna populations on the basis of Nei’s genetic distances. The DNA fragments were visualized under UV light and further analyzed with a video image analyzer (BioImaging Systems, Cambridge, UK). 3) Extraction of plant material and qualitative analysis of alkaloids. From GC-MS Analysis of alkaloids identified from Alkanna primuliflora, A. graeca and A. stribrnyi, chromatograms were used, which were not used in the article. They represent a crude alkaloid mixture of the three species. Based on mass spectral fragmentation with standard reference spectra from the database, alkaloids were identified in all three species

On the Reproductive Potential in Primula veris L. (Primulaceae): Embryological Features, Pollen and Seed Viability, Genetic Diversity

Primula veris (Primulaceae) is a valuable medicinal plant. The main characteristics for assessing the reproductive potential (embryological features; mode of reproduction; pollen and seed viability) and the genetic diversity of populations of the species from Bulgaria were studied. The anthers are tetrasporangiate. Their wall development follows the Dicotyledonous-type and consists of: epidermis, a fibrous endothecium, an ephemeral middle layer and a secretory (glandular) tapetum. After meiosis in pollen mother cells and simultaneous microsporogenesis tetrahedral tetrads are formed predominantly in the anthers. Many ovules (approximately 20) develop in the unilocular ovary and are anatropous, tenuinucellate and bitegmic. The embryo sac (ES) develops after Polygonum (monosporic)-type from the chalazal cell of linear megaspore tetrad in the ovule. After double fertilization, a Caryophyllad-type embryo and initially nuclear endosperm form. In the studied populations, high pollen viability of more than 95% was established. Extremely low viability (about 4%) of the seeds obtained from natural populations was established. The results reveal P. veris to be a predominantly amphimictic (sexually reproducing) species, although rare vegetative propagation is also observed. As a result of this study, essential data were obtained about the reproductive structures and processes and for assessing the reproductive potential of P. veris

On the distribution of Helichrysum arenarium (l.) Moench (Asreraceae) in Bulgaria

Helichrysum arenarium (L.) Moench is herbaceous perennial plant belonging to Asteraceae family. The plant is well known in phytotherapy for its potential in the treatment of gallbladder disease and is classified as endangered in a number of European countries. In Bulgaria it is a protected species according to the Biodiversity Act (Annex 4) and is in the List of Species of Medicinal Plants under special regimen of conservation and use. The data on the distribution of H. arenarium in the Bulgarian flora have not been updated for more than 20 years. The aim of this study is to determine the current distribution of H. arenarium in Bulgaria on the basis of reviewing the available herbarium specimens in the Bulgarian herbariums, literature data, and personal collections. As a result, the locations of the species on the territory of the country were described and mapped. In Bulgaria H. arenarium is represented with only a few populations located in a limited area in the northeastern part of the country, in a narrow strip between the towns of Shumen and Varna

On the Reproductive Potential in <i>Primula veris</i> L. (Primulaceae): Embryological Features, Pollen and Seed Viability, Genetic Diversity

Primula veris (Primulaceae) is a valuable medicinal plant. The main characteristics for assessing the reproductive potential (embryological features; mode of reproduction; pollen and seed viability) and the genetic diversity of populations of the species from Bulgaria were studied. The anthers are tetrasporangiate. Their wall development follows the Dicotyledonous-type and consists of: epidermis, a fibrous endothecium, an ephemeral middle layer and a secretory (glandular) tapetum. After meiosis in pollen mother cells and simultaneous microsporogenesis tetrahedral tetrads are formed predominantly in the anthers. Many ovules (approximately 20) develop in the unilocular ovary and are anatropous, tenuinucellate and bitegmic. The embryo sac (ES) develops after Polygonum (monosporic)-type from the chalazal cell of linear megaspore tetrad in the ovule. After double fertilization, a Caryophyllad-type embryo and initially nuclear endosperm form. In the studied populations, high pollen viability of more than 95% was established. Extremely low viability (about 4%) of the seeds obtained from natural populations was established. The results reveal P. veris to be a predominantly amphimictic (sexually reproducing) species, although rare vegetative propagation is also observed. As a result of this study, essential data were obtained about the reproductive structures and processes and for assessing the reproductive potential of P. veris

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

Chemical Profile and Antimicrobial Activity of the Essential Oils of <i>Helichrysum arenarium</i> (L.) Moench. and <i>Helichrysum italicum</i> (Roth.) G. Don

This study compared the essential oils (EO) composition of Helichrysum arenarium (Bulgarian populations) with that of the cultivated species H. italicum. The EO composition of H. arenarium and H. italicum were analyzed via gas chromatography. In general, 75 components were identified in H. arenarium EO and 79 in H. italicum EO. The predominant constituents in H. arenarium EO were α-pinene (34.64–44.35%) and sabinene (10.63–11.1%), which affirmed the examined population as a new chemical type. Overall, the main EO constituents of H. italicum originating in France, Bosnia and Corsica were neryl acetate (4.04–14.87%) and β-himachalene (9.9–10.99%). However, the EOs profile of H. italicum introduced from the above three countries differed to some extent. D-limonene (5.23%), italicene, α-guaiene and neryl acetate (14.87%) predominated in the H. italicum introduced from France, while α-pinene (13.74%), δ-cadinene (5.51%), α-cadinene (3.3%), β-caryophyllene (3.65%) and α-calacorene (1.63%) predominated in plants introduced from Bosnia. The EOs of the plants introduced from France and Corsica had similar chemical composition and antimicrobiological activity

Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species.

The Balkans endemic species Alkanna primuliflora Griseb., A. stribrnyi Velen., and A. graeca Boiss. & Spruner have limited distribution in the Balkan Peninsula and a large variation in the morphological characteristics. The populations of the three Alkanna species in the Bulgarian flora are small and fragmented. There are no previous reports on the chemical profile or on the embryology of these species. The hypothesis was that the limited distribution of A. primuliflora, A. stribrnyi, and A. graeca was due to their reproductive capacity and genetic diversity. Furthermore, we hypothesized that the three species will contain pyrrolizidine alkaloids (PAs), as other species of the genus Alkanna (Boraginaceae), but they would have differential alkaloids composition. The population genetic structure and differentiation showed a clear distinction between species and revealed average levels of genetic diversity among the natural populations of the three Alkanna species. The embryological investigation observed stability of the processes in the male and female generative spheres and high viability of mature pollen and embryo of the three species. The normal formation of male and female gametophytes without deviations or degenerative processes, and observed levels of genetic diversity between Alkanna individuals are important in maintaining the size and resilience of the Alkanna populations. Eight alkaloids were identified by GC-MS in A. primuliflora and A. graeca and six alkaloids in A. stribrnyi. The main pyrrolizidine alkaloids (PAs) in all investigated species was triangularine. A. primuliflora and A. graeca showed similar chemical composition that comprised 9-angeloylretronecine, 7-tigloylretronecine, 9-tigloylretronecine, triangularicine, dihydroxytriangularine, dihydroxytriangularicine, whereas, in A. stribrnyi 9-tigloylretronecine, triangularicine and dihydroxytriangularicine were not found. This is the first report on the presence of PAs in A. primuliflora, A. stribrnyi and A. graeca. Besides, this is the first report on the embryology of these endemic species. The results contribute to the knowledge of the three endemic Alkanna species and will facilitate policy-making and defining new strategies for their conservation

Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of <i>Alkanna tinctoria</i>

Alkanna tinctoria (L.) Tausch Boraginaceae is a medicinal plant whose root is used for its antimicrobial and anti-inflammatory properties. A. tinctoria roots have been subject to numerous studies. However, the aerial parts have been explored less. The objective of the present study was to compare the chemical profile of aerial parts and roots as well as the total alkannin content in roots of 11 populations of the species from different floristic regions of Bulgaria. Methanolic extracts from 22 samples were analyzed by GC/MS. Phenolic, fatty, and organic acids, sterols, polyols, fatty alcohols, and sugars were identified. Ononitol (4-O-methyl-myo-inositol) was found as the main compound in the aerial parts. The total alkannin content in the roots was evaluated by the spectrophotometric method and compared with that of the commercial product. Populations with high alkannin content and rich in other bioactive compounds were identified. A relatively low genetic diversity in the studied populations was observed. The present study is the first comprehensive study on metabolite profiles and genetic diversity of the Bulgarian populations of A. tinctoria. The occurrence of ononitol in the aerial parts of the species is reported for the first time, as well as the phenolic acid profiles of the species in both aerial parts and roots. The results showed that aerial parts of the plant are also promising for use as a source of valuable biologically active substances

Phytochemical Investigation and Reproductive Capacity of the Bulgarian Endemic Plant Species Marrubium friwaldskyanum Boiss. (Lamiaceae)

Marrubium friwaldskyanum Boiss (Lamiaceae) is a Bulgarian endemic species. Overall, the essential oil (EO) composition of M. friwaldskyanum was different from that of the other Marrubium species reported in the literature. The main EO constituents of M. friwaldskyanum were (E)-caryophyllene, germacrene D, and caryophyllene oxide. The effect of the harvest stage was significant only on &alpha;-copaene, (E)-caryophyllene, caryophyllene oxide, and &tau;-muurolol. The concentration of &alpha;-copaene (1.26&ndash;1.83% range of the total oil), (E)-caryophyllene (31&ndash;41%), caryophyllene oxide (6.4&ndash;11.8%), and &tau;-muurolol (1.3&ndash;2.8%) were the highest at 2&ndash;3 pair of leaves or before flowering and lower at flowering. The harvest stage did not significantly affect the concentrations of the other six identified EO compounds &beta;-bourbonene (1.1%), &alpha;-humulene (2.8%), germacrene D (23.3%), bicyclogermacrene (2.85%), &delta;-cadinene (1.1%), and spathulenol (2.8%). In a separate experiment, grinding of the biomass prior to EO extraction had a significant effect only on the concentrations of D-limonene (0.24&ndash;3.3%) and bicyclogermacrene (3.6&ndash;9.1%). Grinding in water or without water, maceration, and addition of Tween&reg;20 had rather small effects on the EO profile. The identified EO constituents and their mean concentrations in this experiment were (E)-caryophyllene (25.4%), germacrene D (17.6%), caryophyllene oxide (9.1%), spathulenol (6.5%), &tau;-muurolol (5.0%), carvacrol (3.9%), &alpha;-copaene (2.5%), &beta;-bourbonene (2.5%), &delta;-cadinene (2.4%), &alpha;-humulene (1.8%), and Z-&beta;-farnesene (1.3%). Embryological studies observed anther and the development of the male gametophyte and ovule and development of the female gametophyte of M. friwaldskyanum. Furthermore, pollen and seed viability assays were conducted, and mass spectrometry-based metabolomics analysis of an extract from shoots revealed the presence of 45 natural products, identified as flavonoids, phenolic acids, and (tri)terpenoids. Overall, the phytochemistry and some of the microscopic analyses distinguished this endemic species from other species in Marrubium