Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Early selection of carrot somatic hybrids: a promising tool for species with high regenerative ability.

BACKGROUND: Since its discovery, somatic hybridization has been used to overcome the sexual barriers between cultivated and wild species. A combination of two somatic cells might provide a novel set of features, often of agronomical importance. Here, we report a successful approach for production and selection of interspecific somatic hybrid plants between cultivated and wild carrot using dual-labelling of protoplasts and an early selection of fused cells via micromanipulator. Both subspecies used in this study are characterised by a very high regenerative ability in protoplast cultures. Thus, a precise and effective method of hybrid selection is essential to assure the development and regeneration of much less numerous heterokaryons in the post-fusion cell mixture. RESULTS: Electrofusion parameters, such as alternating current and direct current, were optimised for an efficient alignment of protoplasts and reversible membrane breakdown followed by a cell fusion. Four hundred twenty-nine cells emitting green-red fluorescence, identified as hybrids, were obtained. Co-culture with donor-derived protoplasts in the alginate feeder layer system stimulated re-synthesis of the cell wall and promoted cell divisions of fusants. Somatic embryogenesis occurred in hybrid-derived microcalli cultures, followed by plant regeneration. Regenerated hybrids produced yellowish storage roots and leaves of an intermediate shape between cultivated and wild subspecies. The intron length polymorphism analysis revealed that 123 of 124 regenerated plants were hybrids. CONCLUSIONS: The developed protocol for protoplast fusion and an early selection of hybrids may serve as an alternative to combining genomes and transferring nuclear or cytoplasmatic traits from wild Daucus species to cultivated carrot.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn.

BACKGROUND: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. RESULTS: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. CONCLUSIONS: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.

Combining genome size and pollen morphology data to study species relationships in the genus Daucus (Apiaceae).

BACKGROUND: The genus Daucus (Apiaceae) comprises about 40 wild species and the cultivated carrot, a crop of great economic and nutritional importance. The rich genetic diversity of wild Daucus species 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 wild Daucus species. To broaden such knowledge, in this research, the nuclear DNA content for 14 Daucus accessions 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 among Daucus taxa, ranging from 0.999 (D. carota subsp. sativus) to 3.228 pg (D. littoralis). Among the outgroup species, the mean 2C values were 1.775-2.882 pg. The pollen grains of Daucus were 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 in Daucus and provides comprehensive knowledge of the pollen morphology of its taxa. These findings may be important in elucidating the taxonomic relationships among Daucus species 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.

Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.

KEY MESSAGE: We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.

Using carrot centromeric repeats to study karyotype relationships in the genus Daucus (Apiaceae).

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 genus Daucus seems 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, Daucus species 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 other Daucus species, detailed karyomorphological research has been limited to carrot and only a few wild species. Therefore, to better understand the karyotype relationships within Daucus, we (1) explored the chromosomal distribution of carrot centromeric repeats (CentDc) in 34 accessions of Daucus and 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 of Daucus (belonging to both Daucus I 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 other Daucus taxa, 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 both Daucus subclades 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 genus Daucus and may contribute to a better understanding of the dynamic evolution of centromeric satellites in plants.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria.

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

Light Microscopy and Raman Imaging of Carotenoids in Plant Cells In Situ and in Released Carotene Crystals.

Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations, carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for subsequent observations using light microscopy and for analysis using Raman spectroscopy.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae).

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Chiral Amplification in Nature: Studying Cell-Extracted Chiral Carotenoid Microcrystals via the Resonance Raman Optical Activity of Model Systems.

Carotenoid microcrystals, extracted from cells of carrot roots and consisting of 95 % of achiral ß-carotene, exhibit a very intense chiroptical (ECD and ROA) signal. The preferential chirality of crystalline aggregates that consist mostly of achiral building blocks is a newly observed phenomenon in nature, and may be related to asymmetric information transfer from the chiral seeds (small amount of α-carotene or lutein) present in carrot cells. To confirm this hypothesis, we synthesized several model aggregates from various achiral and chiral carotenoids. Because of the sergeant-and-soldier behavior, a small number of chiral sergeants (α-carotene or astaxanthin) force the achiral soldier molecules (ß- or 11,11'-[D2 ]-ß-carotene) to jointly form supramolecular assemblies of induced chirality. The chiral amplification observed in these model systems confirmed that chiral microcrystals appearing in nature might consist predominantly of achiral building blocks and their supramolecular chirality might result from the co-crystallization of chiral and achiral analogues.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.

Characterization of a Genomic Region under Selection in Cultivated Carrot (Daucus carota subsp. sativus) Reveals a Candidate Domestication Gene.

Carrot is one of the most important vegetables worldwide, owing to its capability to develop fleshy, highly nutritious storage roots. It was domesticated ca. 1,100 years ago in Central Asia. No systematic knowledge about the molecular mechanisms involved in the domestication syndrome in carrot are available, however, the ability to form a storage root is undoubtedly the essential transition from the wild Daucus carota to the cultivated carrot. Here, we expand on the results of a previous study which identified a polymorphism showing a significant signature for selection upon domestication. We mapped the region under selection to the distal portion of the long arm of carrot chromosome 2, confirmed that it had been selected, as reflected in both the lower nucleotide diversity in the cultivated gene pool, as compared to the wild (πw/πc = 7.4 vs. 1.06 for the whole genome), and the high FST (0.52 vs. 0.12 for the whole genome). We delimited the region to ca. 37 kb in length and identified a candidate domestication syndrome gene carrying three non-synonymous single nucleotide polymorphisms and one indel systematically differentiating the wild and the cultivated accessions. This gene, DcAHLc1, belongs to the AT-hook motif nuclear localized (AHL) family of plant regulatory genes which are involved in the regulation of organ development, including root tissue patterning. AHL genes work through direct interactions with other AHL family proteins and a range of other proteins that require intercellular protein movement. Based on QTL data on root thickening we speculate that DcAHLc1 might be involved in the development of the carrot storage root, as the localization of the gene overlapped with one of the QTLs. According to haplotype information we propose that the 'cultivated' variant of DcAHLc1 has been selected from wild Central Asian carrot populations upon domestication and it is highly predominant in the western cultivated carrot gene pool. However, some primitive eastern landraces and the derived B7262 purple inbred line still carry the 'wild' variant, reflecting a likely complexity of the genetic determination of the formation of carrot storage roots.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution.

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Composition and (in)homogeneity of carotenoid crystals in carrot cells revealed by high resolution Raman imaging.

Three categories of roots differing in both ß/α-carotene ratio and in total carotenoid content were selected based on HPLC measurements: high α- and ß-carotene (HαHß), low α- and high ß-carotene (LαHß), and low α- and low ß-carotene (LαLß). Single carotenoid crystals present in the root cells were directly measured using high resolution Raman imaging technique with 532nm and 488nm lasers without compound extraction. Crystals of the HαHß root had complex composition and consisted of ß-carotene accompanied by α-carotene. In the LαHß and LαLß roots, measurements using 532nm laser indicated the presence of ß-carotene only, but measurements using 488nm laser confirmed co-occurrence of xanthophylls, presumably lutein. Thus the results show that independently on carotenoid composition in the root, carotenoid crystals are composed of more than one compound. Individual spectra extracted from Raman maps every 0.2-1.0µm had similar shapes in the 1500-1550cm(-1) region indicating that different carotenoid molecules were homogeneously distributed in the whole crystal volume. Additionally, amorphous carotenoids were identified and determined as composed of ß-carotene molecules but they had a shifted the ν1 band probably due to the effect of bonding of other plant constituents like proteins or lipids.

Effect of ß-lactam antibiotics on plant regeneration in carrot protoplast cultures.

Protoplasts of three carrot cultivars were isolated from in vitro-grown plantlets by overnight incubation in an enzyme mixture composed of 1% (w/v) cellulase Onozuka R-10 and 0.1% (w/v) pectolyase Y-23. After cell immobilization in modified thin alginate layers, three types of ß-lactam antibiotics (cefotaxime, carbenicillin, or timentin) at five different concentrations (100, 200, 300, 400, or 500 mg L-1) were added to the culture medium. In 20-d-old cultures, a different number of cell colonies had formed and varied on average from 27 to 56% in carbenicillin- and cefotaxime-containing media, respectively. Supplementation of the culture media with antibiotics at concentrations higher than 100 mg L-1 resulted in a decrease in plating efficiency in comparison with the controls. However, from all antibiotic treatments, except carbenicillin at concentrations of 400-500 mg L-1, efficient plant regeneration occurred. For this reason, we believe that cefotaxime and timentin in the concentrations analyzed here may be used in complex in vitro procedures or valuable carrot cultures as a prophylactic agent for prevention against occasional contaminations.

DcSto: carrot Stowaway-like elements are abundant, diverse, and polymorphic.

We investigated nine families of Stowaway-like miniature inverted-repeat transposable elements (MITEs) in the carrot genome, named DcSto1 to DcSto9. All of them were AT-rich and shared a highly conserved 6 bp-long TIR typical for Stowaways. The copy number of DcSto1 elements was estimated as ca. 5,000 per diploid genome. We observed preference for clustered insertions of DcSto and other MITEs. Distribution of DcSto1 hybridization signals revealed presence of DcSto1 clusters within euchromatic regions along all chromosomes. An arrangement of eight regions encompassing DcSto insertion sites, studied in detail, was highly variable among plants representing different populations of Daucus carota. All of these insertions were polymorphic which most likely suggests a very recent mobilization of those elements. Insertions of DcSto near carrot genes and presence of putative promoters, regulatory motifs, and polyA signals within their sequences might suggest a possible involvement of DcSto in the regulation of gene expression.

Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

Paternity determination of interspecific rhododendron hybrids by genomic in situ hybridization (GISH).

Genomic in situ hybridization (GISH) has been proved to be the most effective and accurate technique for confirmation of hybrid character. The objective of our study was to adapt and optimize a GISH protocol for identification of donor chromatin in hybrids obtained by interspecific crosses between five Rhododendron taxa (R. aureum, R. brachycarpum, R. catawbiense 'Catharine van Tol', R. catawbiense 'Nova Zembla', and R. yakushimanum 'Koichiro Wada'). Positive results were obtained only when we used mitotic chromosome spreads prepared from anthers. The best differentiation of maternal and paternal chromosomes in hybrid genomes was obtained when 50 ng of probe was applied together with blocking DNA at a concentration of 3.0 microg/microL. The results demonstrate that GISH is a practical tool for detection of alien genomes and analysis of the constitution of the chromosomes in rhododendron hybrids.