Genetic diversity and population structure are key resources for breeding purposes and genetic studies of important agronomic traits in crops. In this study, we described SNP-based genetic diversity, linkage disequilibrium and population structure in a panel of 179 bread wheat advanced cultivars and old accessions from Bulgaria, using an optimized wheat 25K Infinium iSelect array. Out of 19,019 polymorphic SNPs, 17,968 had a known chromosome position on the A (41%), B (42%) and D (11%) genome, and 6% were not assigned to any chromosome. Homoeologous group 4, in particular chromosome 4D, was the least polymorphic. In the total population, the Nei’s gene diversity was within the range 0.1–0.5, and the polymorphism information content ranged from 0.1 to 0.4. Significant differences between the old and modern collections were revealed with respect to the linkage disequilibrium (LD): the average values for LD (r²), the percentage of the locus pairs in LD and the LD decay were 0.64, 16% and 3.3 for the old germplasm, and 0.43, 30% and 4.1 for the modern releases, respectively. Structure and k-means clustering algorithm divided the panel into three groups. The old accessions formed a distinct subpopulation. The cluster analysis further distinguished the modern releases according to the geographic region and genealogy. Gene exchange was evidenced mainly between the subpopulations of contemporary cultivars. The achieved understanding of the genetic diversity and structure of the Bulgarian wheat population and distinctiveness of the old germplasm could be of interest for breeders developing cultivars with improved characteristics. The obtained knowledge about SNP informativeness and the LD estimation are worthwhile for selecting markers and for considering the composition of a population in association mapping studies of traits of interest.

At present, more than 50 000 plant species are used in phytotherapy and medicine. About 2/3 of them are harvested from nature leading to local extinction of many species or degradation of their habitats. Biotechnological methods offer possibilities not only for faster cloning and conservation of the genotype of the plants but for modification of their gene information, regulation, and expression for production of valuable substances in higher amounts or with better properties.Rhodiola roseais an endangered medicinal species with limited distribution. It has outstanding importance for pharmaceutical industry for prevention and cure of cancer, heart and nervous system diseases, and so forth. Despite the great interest in golden root and the wide investigations in the area of phytochemistry, plant biotechnology remained less endeavoured and exploited. The paper presents research on initiation ofin vitrocultures inRhodiola roseaand some otherRhodiola species. Achievements in induction of organogenic and callus cultures, regeneration, and micropropagation varied but were a good basis for alternativein vitrosynthesis of the desired metabolites and for the development of efficient systems for micropropagation for conservation of the species.

Clinopodium vulgare L. is a valuable medicinal plant used for its anti-inflammatory, antibacterial and wound-healing properties. The present study describes an efficient protocol for the micropropagation of C. vulgare and compares, for the first time, the chemical content and composition and antitumor and antioxidant activities of extracts from in vitro cultivated and wild-growing plants. The best nutrient medium was found to be Murashige and Skoog (MS) supplemented with 1 mg/L BAP and 0.1 IBA mg/L, yielding on average 6.9 shoots per nodal segment. Flower aqueous extracts from in vitro plants had higher total polyphenol content (29,927.6 ± 592.1 mg/100 g vs. 27,292.8 ± 85.3 mg/100 g) and ORAC antioxidant activity (7281.3 ± 82.9 µmol TE/g vs. 7246.3 ± 62.4 µmol TE/g) compared to the flowers of wild plants. HPLC detected qualitative and quantitative differences in phenolic constituents between the in vitro cultivated and wild-growing plants’ extracts. Rosmarinic acid was the major phenolic constituent, being accumulated mainly in leaves, while neochlorogenic acid was a major compound in the flowers of cultivated plants. Catechin was found only in cultivated plants, but not in wild plants or cultivated plants’ stems. Aqueous extracts of both cultivated and wild plants showed significant in vitro antitumor activity against human HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) cell lines. The best cytotoxic activity against most of the cancer cell lines, combined with the least detrimental effects on a non-tumor human keratinocyte cell line (HaCaT), was shown by the leaf (250 µg/mL) and flower (500 µg/mL) extracts of cultivated plants, making cultivated plants a valuable source of bioactive compounds and a suitable candidate for anticancer therapy.

Sideritis scardica Griseb. is a critically endangered Balkan endemic species, known for its antioxidant, neuroprotective and anti-inflammatory properties. The aim of the present study was to detail an efficient protocol for the micropropagation of S. scardica. In vitro cultures were initiated from the shoot tips of 40 days-old in vivo seedlings and the effects of different plant growth regulator treatments were examined. A Murashige and Skoog nutrient medium (MS) containing 1 mg/L zeatin and 0.1 mg/L indole-3-acetic acid (IAA) proved to be the most efficient for shoot multiplication as it produced quality, vigorous shoots with a mean number of six shoots per explant. For the first time, the antioxidant and antitumor activities of extracts from in vitro-obtained plants were evaluated. In vitro cultivated plants grown in the field revealed a higher total polyphenol content (3929.1 ± 112.2 mg GAE/100 g vs. 3563.5 ± 52.8 mg GAE/100 g) and higher ORAC antioxidant activity (1211.6 ± 27.3 µmol TE/g vs. 939.9 ± 52.4 µmol TE/g) than in situ cultivated plants. A comparison of the antitumor activities of extracts from in vitro propagated shoots, field-grown in vitro-obtained plants and in situ plants on HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) human cancer cell lines showed that in vitro propagated shoots had a significant concentration-dependent cytotoxic effect on the cervical adenocarcinoma cell line HeLa, while the field-grown in vitro-obtained and in situ-collected samples induced the highest reduction in the viability of the mammary carcinoma cell line MCF-7. In both cases, the cells of the control non-tumor cell line, BALB/3T3, were significantly less affected. The results showed that the in vitro multiplication protocol ensured the obtainment of numerous plants with antioxidant and antitumor potential.

The potential of Marrubium vulgare to alleviate scopolamine (Sco)-induced deficits in spatial working memory has drawn considerable scientific interest. This effect is partly attributed to its potent antioxidant and acetylcholinesterase inhibitory (AChEI) activities. This study examined the effects of M. vulgare extract, standardized to marrubiin content, on recognition memory in healthy and Sco-treated rats. Male Wistar rats (200–250 g) were divided into four groups. The extract was orally administered for 21 days and Sco (2 mg/kg) was intraperitoneally injected for 11 consecutive days. Memory performance was assessed using the novel object recognition test. Levels of acetylcholine (ACh), noradrenaline (NA), serotonin (Sero), and brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element-binding protein (p-CREB) were evaluated in the cortex and hippocampus via ELISA. BDNF and CREB expression levels were assessed using RT-PCR. The results showed that M. vulgare significantly alleviated Sco-induced memory impairment, preserved cholinergic function in the hippocampus, increased NA levels in the brain, and restored pCREB expression in the cortex following Sco-induced reduction. In healthy rats, the extract upregulated BDNF, pCREB, and Bcl2 expression. Our findings indicate that the neuroprotective effects of M. vulgare may be linked to the modulation of cholinergic function, regulation of NA neurotransmission, and influence on key memory-related molecules.

Rhodiola rosea is an endangered medicinal plant used for cancer, cardiovascular, and nervous system diseases therapy. Due to its limited distribution and sustainability alternative methods for production of its valuable substances are under investigation. Using in vitro techniques apical and rhizome buds, leaf nodes, stem and radix segments from wild plants and in vitro seedlings were plated on 24 modified Murashige and Skoog (1962) media. Decontamination of plant material was successful only in 21% of the schemes. The best shoot induction was obtained from seedling explants on media containing 2 mg/l zeatin or N6-benzylaminopurine, each. Their reduction stimulated shoot formation in the next passages (multiplication rate up to 5). Efficient rooting was induced on half-strength MS with 2 mg/l Indole-3-butyric acid and stimulated by adding 0.2 mg/l Indolyl-3-acetic acid. Regenerants rooted in perlite, peat, and soil (1:1:2), adapted in greenhouse, and transplanted in the mountains survived (70%) and developed like the wild plants. Salidroside content of these plants after one or two years was high (0.64 and 0.61% in rhizomes and 0.62 and 0.53% in roots, respectively). This is the first established efficient scheme for micropropagation of Bulgarian R. rosea allowing habitats restoration, germplasm conservation, and potential application of biotechnology for production of valuable substances.