In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L⁻¹ 6-benzylaminopurine and 0.5 mg L⁻¹α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.

Background: The genusDaucus(Apiaceae) comprises about 40 wild species and the cultivated carrot, a crop of great economic and nutritional importance. The rich genetic diversity of wildDaucusspecies makes them a valuable gene pool for carrot improvement breeding programs. Therefore, it is essential to have good knowledge of the genome structure and relationships among wildDaucusspecies. To broaden such knowledge, in this research, the nuclear DNA content for 14Daucusaccessions and four closely related species was estimated by flow cytometry and their pollen morphology was analyzed by light and scanning electron microscopy (SEM).Results: The flow cytometric analysis showed a 3.2-fold variation in the mean 2C values amongDaucustaxa, ranging from 0.999 (D.carotasubsp.sativus) to 3.228 pg (D.littoralis). Among the outgroup species, the mean 2C values were 1.775–2.882 pg. The pollen grains ofDaucuswere tricolporate, mainly prolate or perprolate (rarely) in shape, and mainly medium or small (rarely) in size (21.19–40.38 µm), whereas the outgroup species had tricolporate, perprolate-shaped, and medium-sized (26.01–49.86 µm) pollen grains. In the studied taxa, SEM analysis revealed that exine ornamentation was striate, rugulate, perforate, or the ornamentation pattern was mixed. At the time of shedding, all pollen grains were three-celled, as evidenced by DAPI staining. We also found high positive correlations between the length of the polar axis (P) and the length of the equatorial diameter (E) of pollen grains, as well as between P and P/E. However, when comparing cytogenetic information with palynological data, no significant correlations were observed.Conclusions: This study complements the information on the nuclear DNA content inDaucusand provides comprehensive knowledge of the pollen morphology of its taxa. These findings may be important in elucidating the taxonomic relationships amongDaucusspecies and can help in the correct identification of gene bank accessions. In a broader view, they could also be meaningful for the interpretation of evolutionary trends in the genus.

Background: In the course of evolution, chromosomes undergo evolutionary changes; thus, karyotypes may differ considerably among groups of organisms, even within closely related taxa. The genusDaucusseems to be a promising model for exploring the dynamics of karyotype evolution. It comprises some 40 wild species and the cultivated carrot, a crop of great economic significance. However,Daucusspecies are very diverse morphologically and genetically, and despite extensive research, the taxonomic and phylogenetic relationships between them have still not been fully resolved. Although several molecular cytogenetic studies have been conducted to investigate the chromosomal structure and karyotype evolution of carrot and otherDaucusspecies, detailed karyomorphological research has been limited to carrot and only a few wild species. Therefore, to better understand the karyotype relationships withinDaucus, we (1) explored the chromosomal distribution of carrot centromeric repeats (CentDc) in 34 accessions ofDaucusand related species by means of fluorescence in situ hybridization (FISH) and (2) performed detailed karyomorphological analysis in 16 of them.Results: We determined the genomic organization of CentDc in 26 accessions ofDaucus(belonging to bothDaucusI and II subclades) and one accession of closely related species. The CentDc repeats were present in the centromeric regions of all chromosomes of 20 accessions (representing 11 taxa). In the otherDaucustaxa, the number of chromosome pairs with CentDc signals varied depending on the species, yet their centromeric localization was conserved. In addition, precise chromosome measurements performed in 16 accessions showed the inter- and intraspecific karyological relationships among them.Conclusions: The presence of the CentDc repeats in the genomes of taxa belonging to bothDaucussubclades and one outgroup species indicated the ancestral status of the repeat. The results of our study provide useful information for further evolutionary, cytotaxonomic, and phylogenetic research on the genusDaucusand may contribute to a better understanding of the dynamic evolution of centromeric satellites in plants.

Fruit morphological and anatomical characteristics are essential in the taxonomy of Apiaceae. Daucus L. is one of the most important genera of the family Apiaceae, as it contains the cultivated carrot, a crop of great economic importance, and about 40 wild species that could serve as potential sources of genetic diversity for crop improvement. However, the taxonomic and phylogenetic relationships among these species have not yet been fully clarified. In this study, we comparatively investigated the fruit morphology and anatomy of 13 Daucus taxa and four closely related non-Daucus species using light and scanning electron microscopy to evaluate the taxonomic value of these characteristics. A wide range of variations was observed in the fruit morpho-anatomical characteristics across the taxa and revealed several diagnostically valuable features, thus proving to be taxonomically useful. For Daucus, the observed differences included the fruit size (2.1–8.4 mm), shape (from ellipsoid to oblong), and weight (0.079–1.349 g/100 fruits), as well as the fruit surface sculpturing and some anatomical characteristics, i.e., the presence/absence and size of vittae, the shape and size of vascular bundles, and the shape of exocarp cells. This study contributes to a better understanding of the relationships among the genus Daucus.

Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet (Beta vulgaris L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages. We established a methyl salicylate-based protocol that significantly improved the transparency of the B. vulgaris ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat. The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species’ embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.