Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Genetic Diversity and Population Structure of Bulgarian Autochthonous Sheep Breeds Revealed by Microsatellite Analysis.

This study attempts to provide a deeper insight into the current genetic status of 12 Bulgarian autochthonous sheep breeds using microsatellite (SSR) markers. A total of 600 individuals from 50 flocks were analyzed using a panel of 13 SSR markers. In total, 228 alleles were found in the studied microsatellite loci. The mean number of alleles, the effective number of alleles, and the polymorphic information content (PIC) values per locus were 17.54, 5.250, and 0.799, respectively. The expected heterozygosity (He) for all breeds ranged from 0.70 to 0.82. The within-population heterozygote deficit (Fis) varied from -0.03 to 0.1, reflecting significant levels for 10 of the 12 breeds. The average genetic differentiation (Fst) was 0.046, revealing a low discrimination between the breeds. The genetic distance, principal coordinate analysis, and the structure analysis showed that two of the studied breeds-Local Stara Zagora/SZ/ and Local Karnobat/MK/-were the most distinct sheep populations. The Bayesian clustering approach suggested poor breed differentiation for the remaining 10 sheep breeds. The results suggest that proper management strategies and specific breeding policies need to be implemented in Bulgaria to avoid the intermixing of breeds and to reduce the erosion of breed purity observed in some breeds.

A Set of Highly Polymorphic Microsatellite Markers for Genetic Diversity Studies in the Genus Origanum.

This study reports the development of a set of 20 highly polymorphic genomic SSR markers which can be used for both cultivar identification and genetic diversity studies in several Origanum species, including some of the most popular ones like Greek oregano (Origanum vulgare L. ssp. hirtum), common oregano (O. vulgare L. ssp. vulgare), and sweet marjoram (O. majorana L.). Analysis of the polymorphic information content (PIC) showed an average PIC value of 0.75 with a minimum of 0.41 and a maximum of 0.89, where 17 of the markers showed PIC values above 0.73. Comparative analysis of the genetic diversity of eight natural populations of Greek oregano in Bulgaria showed that six of the genomic SSR markers revealed significantly higher portions of genetic diversity in the populations, compared to 12 EST SSR markers used in our previous study. We also compared the performance of the same six genomic SSR markers with the results for eight SRAP primer combinations, which showed that SRAP markers captured more precisely the genetic structure in natural populations. The developed highly polymorphic genomic SSR markers can be successfully applied to evaluation of the genetic diversity in the genus Origanum, based on the expected and observed heterozygosity in the populations as well as for easy identification of breeding lines and cultivars based on unique SSR fingerprints.

Indigenous Yeasts from Rose Oil Distillation Wastewater and Their Capacity for Biotransformation of Phenolics.

The indigenous yeasts associated with the spontaneous fermentation of phenolic-rich rose oil distillation wastewater (RODW) generated after the industrial distillation of rose oil were studied. The ITS-rDNA sequence analysis of the samples collected from RODW fermented at semi-sterile conditions, a waste deposition lagoon and endophytic yeasts isolated from industrially cultivated Rosa damascena suggests that the spontaneous RODW fermentation is caused by yeasts from the genus Cyberlindnera found also as endophytes in the rose flowers. Phylogenetic analysis based on the nucleotide sequences of the translation elongation factor (TEF1α) and 18S- and 26S- rRNA genes further confirmed the taxonomic affiliation of the RODW yeast isolates with the genus Cyberlindnera. The RODW fermentation capacity of a selected set of indigenous yeast isolates was studied and compared with those of common yeast strains. The indigenous yeast isolates demonstrated a superior growth rate, resulting in a nearly double reduction in the phenolic content in the fermented RODW. The indigenous yeasts' fermentation changed the RODW phenolics' composition. The levels of some particular phenolic glycosides decreased through the depletion and fermentation of their sugar moiety. Hence, the relative abundance of the corresponding aglycons and other phenolic compounds increased. The capacity for the biotransformation of RODW phenolics by indigenous yeasts is discussed.

Genetic and Flower Volatile Diversity in Natural Populations of Origanum vulgare subsp. hirtum (Link) Ietsw. in Bulgaria: Toward the Development of a Core Collection.

We studied the genetic and flower volatile diversity in natural populations of Origanum vulgare subsp. hirtum (Link) Ietsw. in Bulgaria using simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) markers and gas chromatography/mass spectrometry (GC/MS) analysis of flower volatiles from individual plants. Two regions, including the Kresna Gorge and Eastern Rhodopes, typical for the species comprising eight populations and 239 individual plants were included in this study. An analysis with 11 SSR markers and eight SRAP primer combinations showed that SRAP markers were substantially more informative than the SSR markers and were further used for genetic diversity analysis. The results showed low-range to mid-range genetic differentiation between the populations with pairwise fixation index (Fst) values ranging between 0.0047 and 0.11. A total of 10 genetic clusters were identified. An analysis of the flower volatile diversity identified a total of 63 compounds with the vast majority of plants belonging to the carvacrol chemotype and just a single plant to the thymol chemotype. Large deviations were observed for individual compounds within each region as well as within the populations. Hierarchical clustering showed a clear sample grouping based on the two different regions. In addition, an in-depth analysis identified six major and 23 minor metabolite clusters. The overall data set and cluster analysis were further used for the development and testing of a simple and straightforward strategy for the selection of individual plants for the development of a core collection representing the sampled natural populations for this species in Bulgaria. The proposed strategy involves precise genetic clustering of the tested plants followed by the selection of a minimal set from each genetic cluster representing the different metabolite clusters. The selected core set was further compared with a core set extracted by the PowerCore software. A comparison of the genetic and metabolic affiliation of the members of both sets showed that the reported approach selected representatives from each genetic cluster and minor metabolic cluster, whereas some metabolic clusters were unrepresented in the PowerCore set. The feasibility and efficiency of applying the pointed strategy for the development of a core collection representing both the genetic and metabolite diversity of natural populations in aromatic and medicinal plants toward subsequent steps of selection and breeding are discussed.

Recovery of polyphenols from rose oil distillation wastewater using adsorption resins--a pilot study.

The production of rose oil from rose flowers by water steam distillation leaves a water fraction of the distillate as main part of the waste. Therefore, the rose oil distillation wastewater represents a serious environmental problem due to the high content of polyphenols which are difficult to decompose and have to be considered as biopollutants when discarded into the drainage system and rivers. On the other hand, natural polyphenols are valuable compounds with useful properties as bioactive substances. Until now there is no established practice for processing of rose oil distillation wastewater and utilization of contained substances. Thus, it was the aim of this study to develop a strategy to separate this wastewater into a polyphenol depleted water fraction and a polyphenol enriched fraction which could be developed into innovative value-added products. In a first step, the phytochemical profile of rose oil distillation wastewater was determined. Its HPLC-PDA-MS analysis revealed the presence of flavan-3-ols, flavanones, flavonols and flavones. In a second step, the development of a stepwise concentration of rose oil distillation wastewater was performed. The concentration process includes a filtration process to eliminate suspended solids in the wastewater, followed by adsorption of the contained phenolic compounds onto adsorption resins (XAD and SP). Finally, desorption of the polyphenol fraction from the resin matrix was achieved using ethanol and/or aqueous ethanol. The result of the process was a wastewater low in soluble organic compounds and an enriched polyphenol fraction (RF20 SP-207). The profile of this fraction was similar to that of rose oil distillation wastewater and showed the presence of flavonols such as quercetin and kaempferol glycosides as major metabolites. These compounds were isolated from the enriched polyphenol fraction and their structures confirmed by NMR. In summary, a pilot medium scale system was developed using adsorption resins for the recovery of polyphenols from rose oil distillation wastewater suggesting an industrial scalability of the process.