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An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 10⁶ and 10⁶ protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.

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.

Protoplasts of four Daucus carota subspecies and three wild Daucus species were isolated from 2-week-old shoot cultures during overnight incubation in an enzyme mixture composed of 1 % (w/v) cellulase Onozuka R-10 and 0.1 % (w/v) pectolyase Y-23. Before the culture, they were embedded in autoclave- or filter-sterilized alginate solution. Modified thin alginate layer (TAL) and extra thin alginate film (ETAF) techniques were applied for protoplast immobilization. A rich mineral-organic medium based on the formulation of Kao and Michayluk supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid, 0.2 mg l⁻¹ zeatin, and optionally 100 nM phytosulfokine (PSK), a peptidyl plant growth factor, was used for protoplast culture. Plating efficiency was genotype-dependent and in 40-day-old cultures, it varied from 10 % for Daucus pusillus to 73 % for D. carota subsp. sativus. A considerably higher ability in colony formation was observed in the modified TAL culture system using filter-sterilized alginate and in the presence of PSK in the protoplast culture medium. Plant regeneration through somatic embryogenesis stimulated by PSK application occurred for five out of the seven Daucus accessions used in the present study. We believe our data may facilitate the use of wild Daucus in somatic hybridization with cultivated carrot.

Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution. Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives. Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci. All had single loci for both 5S and 18S‐25S (nucleolar organizing region) rDNA, located on two different chromosome pairs. The 5S rDNA was on the short arm of a metacentric chromosome pair in D. crinitus (2n = 22) and D. glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome. For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S‐25S rDNA sites. Variability for number and position of the 5S rDNA was also observed. FISH signals enabled us to identify 20–40% of the chromosome complement among species examined. Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus.