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Wild Cucumis species have been divided into Australian/Asian and African groups using morphological and phylogenetic characteristics, and new species have been described recently. No molecular cytogenetic information is available for most of these species. The crossability between 5 southern African Cucumis species (C. africanus, C. anguria, C. myriocarpus, C. zeyheri, and C. heptadactylus) has been reported; however, the evolutionary relationship among them is still unclear. Here, a molecular cytogenetic analysis using FISH with 5S and 45S ribosomal DNA (rDNA) was used to investigate these Cucumis species based on sets of rDNA-bearing chromosomes (rch) types I, II and III. The molecular cytogenetic and phylogenetic results suggested that at least 2 steps of chromosomal rearrangements may have occurred during the evolution of tetraploid C. heptadactylus. In step 1, an additional 45S rDNA site was observed in the chromosome (type III). In particular, C. myriocarpus had a variety of rch sets. Our results suggest that chromosomal rearrangements may have occurred in the 45S rDNA sites. We propose that polyploid evolution occurred in step 2. This study provides insights into the chromosomal characteristics of African Cucumis species and contributes to the understanding of chromosomal evolution in this genus.

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4′,6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram.

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.

A molecular cytogenetic survey of Cucumis sativus L. revealed a chromosomal polymorphism among 2 European pickling cultivars of ‘Borszczagowski’ and ‘Monastyrski’, and one Japanese cultivar of ‘Zibai’ by fluorescent banding method with chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI), and fluorescence in situ hybridization (FISH) with the probes of 5S, 45S rDNA and telomere repeat sequences. Heterochromatic banding polymorphism was found in some chromosomes with CMA and DAPI staining, whereas no chromosomal polymorphism was found in FISH. Thus, the FISH allowed us to identify all of homologous chromosomes in each cucumber cultivars, while CMA and DAPI banding showed an easy detection of the polymorphism in cucumber chromosomes, even in close related cultivars between European pickling cultivars.

Fluorescent in situ hybridisation (FISH) of 5S and 18S-5.8S-26S ribosomal DNA was carried out in two species of the genus Artemisia, belonging to the subgenera Artemisia (A. medioxima) and Absinthium (A. lagocephala), each one showing both low and high ploidy levels (2x, 4x and 16x, and 2x and 6x, respectively). Both species have a base chromosome number of x = 9. Linkage of both rDNA genes has been observed confirming previous results. Diploid A. lagocephala (2n = 18) shows three rDNA loci, and the hexaploid six. Also in A. medioxima, the number of rDNA loci does not increase in the proportion given by the ploidy level, and a relative loss is found. In this species, the diploid population shows two rDNA loci, the tetraploid four, and the hexaidecaploid has around 20. The results evidence a relative loss of rDNA loci and heterochromatin, a phenomenon that is more pronounced at higher ploidy levels. Nevertheless, the DAPI banding pattern of A. lagocephala does not follow this trend, as it shows a spectacular increase of heterochromatic bands at the hexaploid level. These results are discussed in the light of possible chromosome restructuring and gene silencing mechanisms that take place during polyploidy, and more especially allopolyploid formation., This work was subsidized by DGICYT (Spanish Government; projects BOS2001-3041-C02-01, CGL2004-04563-C02-02/BOS, CGL2007-64839-C02-02/BOS and CGL2007-64839-C02-01/BOS). One of the authors (J. P.) has been awarded a predoctoral grant from the Spanish Ministry of Education and Science, and another one (O. H.) benefited from a MICINN (Spanish Ministry of Science and Innovation) postdoctoral grant.

The positions of heterochromatic bands, 45S and 5S rDNA regions were detected on the chromosomes in three cultivars of cucumber (Cucumis sativus L.). The post-heated DAPI bands, which revealed heterochromatic region, were observed on the both terminal and pericentromeric regions of most of the chromosomes in three cultivars. However, these band patterns were different between European and Eastern Asian cultivars. The major 45S rDNA sites were found around the centoromeric region of the chromosomes 1, 2 and 5, and the minor 45S rDNA sites were found around centoromeric region of the chromosomes 3 and 7. The positional variation of this region was not found among three cultivars, but size difference was observed in the chromosome 5. The 5S rDNA site was observed on the interstitial region of the chromosome 6 in all of the cultivars studied. These results indicated that the variation of chromosome structure among cucumber cultivars occurred in the heterochromoatic region and suggested that the genetic variation in cucumber cultivars would be revealed by strict analysis and comparison for the position and size of heterochromatic region.

To resolve the discrepancy among previous karyotype descriptions, chromosome comparative study between two closely related cucumber cultivars, C. sativus L. cv. Borszczagowski and cv. Monastyrski, was performed by chromosome preparation technique with enzymatic maceration, and fluorescent banding method with chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). Heterochromatic band polymorphisms were detected in chromosomes 2, 3, 5 and 7, whereas no chromosomal polymorphism was found in chromosomes 1, 4 and 6. In cv. Borszczagowski chromosome 2, CMA+DAPI+ heterochromatic band at terminal region of the long arm was smaller than that of cv. Monastyrski. Chromosome 3 of cv. Monastyrski was characterized by CMA+DAPI+ bands on the terminal regions of both arms, whereas that of cv. Borszczagowski displayed only on the terminal region of long arm. Distinct CMA+DAPI+ band located at terminal region of the long arm in cv. Borszczagowski was larger than that of cv. Monastyrski. In chromosome 7, terminal CMA+DAPI+ band at the short arm was only observed in cv. Monastyrski. This result suggested that inconsistency of karyotypes among previous reports was due to chromosomal polymorphism.

The chromosome numbers of four species of Ochrosia sensu lato (Apocynaceae) were reported: those of Neisosperma glomerata (2n=22) and Neisosperma citrodora (2n=23) were documented here for the first time, while those of Neisosperma oppositifolia (2n=22) and Ochrosia coccinea (2n=22) verified the previous counts.

Defined changes in the cell wall directed by many proteins accompany every morphogenetic process in plants. Xyloglucan endotransglucosylase/hydrolase proteins (XTH; EC 2.4.1.207) have the potential to modify the hemicellulose matrix within the cell wall. Cs-XTH1 and Cs-XTH3 genes, which encode XTH proteins, were found among numerous genes that are differentially expressed after the induction of cucumber somatic embryogenesis. The expression of these genes increased during somatic embryogenesis. The Cs-XTH1 gene was localized on the second chromosome near the centromere region, whereas Cs-XTH3 was found in the middle of the fifth chromosome's longer arm. Northern blot hybridization showed that both genes were preferentially expressed in roots. We also observed higher accumulation of both transcripts in somatic embryos than in the proembryogenic mass. The localization of mRNA by in situ hybridization revealed that the Cs-XTH1 transcripts were largely accumulated in the presumptive cotyledon primordia of somatic embryos. The XTH gene family consists of a number of genes with a high degree of structural similarity. Screening a cucumber genomic library has identified other members of this gene family. The intron/exon structure, sequence similarities and the close chromosomal distance between some members suggest their common evolutionary origin. The involvement of XTH-related genes in somatic embryo formation is discussed.

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4',6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram.

Zamia angustifolia, Z. integrifolia, Z. pumila and Z. pygmaea had the common chromosome number of 2n= 16 and the common karyotype which consisted of 12 median-, 2 submedianand 2 subterminal-centromeric chromosomes. The 4 species showed commonly the CMA-positive band in the centromeric region of 2 median-, 2 submedian-and 2 subterminal-centromeric chromosomes. In contrast, Z. furfuracea, Z. loddigesii, Z. skinneri and Z. vazquezii had commonly the chromosome number of 2n=18 and 10 median-and 2 submedian-centromeric chromosomes in karyotype but exhibited 6 different chromosomes from each other. Zamia furfuracea and Z. loddigesii displayed the CMA-positive band in the centromeric region of 8 median-and 6 acro-centromeric chromosomes. Zamia skinneri performed the CMA-positive band in the centromeric region of 4 acro-and 2 terminal-centromeric chromosomes, while Z. vazquezii performed the CMA-positive band in the centromeric region of 4 median-and 6 terminal-centromeric chromosomes. Zamia muricata had the chromosome number of 2n=23 differed from that of the previous count, the karyotype which consisted of 7 median-and 16 acro-centromeric chromosomes and the CMA-positive band in the centromeric region of 4 median-and 16 acro-centromeric chromosomes. Then, the 9 species of Zamia studied exhibited unclear DAPI-positive bands near the centromeric region of most of their chromosomes. By comparison, the closely related Ceratozamia mexicana had the chromosome number of 2n=16 and the CMA-positive band at the terminal region of 6 chromosomes.

Effects of the five macro-components and sucrose in half strength MS (1/2MS) agar medium on red-color pigmentation were studied in the plant bodies of Dionaea muscipula and Drosera spathulata generated from multiple shoots in vitro. In 1/2MS agar medium modified with 10.31mM NH4NO3 and 9.40mM KNO3 and supplemented with 0.75 or 0% sucrose the subcultured plants continuously proliferated by multiple shoots and generated large, green-colored plants, while with dilution of those nitrogen components and increase of sucrose to 1.5% the red-color anthocyanin pigmentation spread from the glands or glandular hairs to the entire leaves and the plant sizes and dry weight decreased in inverse proportion to the depth of red color. The anthocyanin pigments of Dionaea muscipula consisted of delphinidin 3-O-glucoside which was new to the species and cyanidin 3-O-glucoside (chrysanthemin), and those of Drosera spathulata, studied here for the first time, consisted of cyanidin 3, 5-di-O-glucoside (cyanin), cyanidin 3-O-galactoside (idaein), cyanidin 3-O-glucoside, pelargonidin 3-O-galactoside, and pelargonidin 3-O-glucoside (callistephin).

Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed.

The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper.

Leaf mesophyll protoplasts of Lycopersicon esculentum were fused with suspension-culture-derived protoplasts of Solanum lycopersicoides by a PEG treatment. Both species have the same chromosome number (2n = 2x = 24). The hybrid calli were selected using the full selection method - kanamycin resistance and culture conditions critical for L. esculentum protoplast divisions. The genomic in situ hybridization analyses indicated a hypo- and hypertetraploid character of the hybrid plant with a majority of S. lycopersicoides chromosomes and a variation in chromosome number from 46 to 53. The hybrids contained a transgene derived from L. esculentum, as shown by Southern blot hybridization and PCR analyses. Their mitochondria were derived from the wild species, S. lycopersicoides. More than 60 regenerated plants were transferred into the greenhouse. They grew very slowly and were not able to flower for almost one year. The main morphological characters of the hybrids included a single shoot and small, dark-green leaves with strongly wrinkled blades. The reasons for nuclear genome asymmetry between hybrids and the possibilities of using them in a genetic and breeding programme are discussed in this paper.

The chromosomal sites of 5S rDNA, 45S rDNA and Arabidopsis-iypt telomere sequence repeats characterize individual species and population constitutions in chrysanthemums and cycads and those of Sau3A in spruces. The sites of the telomere sequence repeats in the cycad chromosomes occur not only in the usual terminal regions of chromosome-arms as small dots, but also in several centromeric to proximal regions as relatively large signals. Large signals of the 278 bp DNA segment with respect to Cycas revoluta involved with pCrT7-4. The Y-chromosome of Cycas revoluta was distinct by lack of the large signal of pCrT7-4 and the Arabidopsis-type telomere sequence repeats. A highly conserved chromosomal location of these genes lead to speculation on the karyotype evolution and species property of the chrysanthemum and cycad groups.

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar – Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

Somatic embryogenesis (SE) in plants can be used as a model for studying genes engaged in the embryogenic transition of somatic cells. The CsSCARECROW (CsSCR) gene was previously identified among a panel of genes upregulated after the induction of SE in cucumber (Cucumis sativus). The putative CsSCR protein contains conserved GRAS family domains and is extremely similar to AtSCR from Arabidopsis thaliana. SCR proteins are transcription factors involved in root radial patterning and are required for main- tenance of the quiescent centre and differentiation of the endodermis. In comparison with other GRAS proteins from cucumber, phylogenetic analyses showed that CsSCR belongs to the SCR cluster. Increased CsSCR transcript accumulation was detected in somatic embryos and roots. Southern blot analysis and screening of the draft version of the cucumber genome confirmed the lack of close homologues in this spe- cies. CsSCR transcripts were localized by in situ hybridization in undifferentiated cells in the globular and heart stages of somatic embryogenesis, and in the endodermis of torpedo and cotyledonary stage somatic embryos, and developing primary and lateral roots. This localization was supported by the pattern of reporter gene activity driven by the CsSCR promoter in transgenic cucumber organs. These results suggest that CsSCR is likely to act in tissue radial organization during somatic embryogenesis and root development.