Increased soil salinization, tightly related to global warming and drought and exacerbated by intensified irrigation supply, implies highly detrimental effects on staple food crops such as wheat. The situation is particularly alarming for durum wheat (DW), better adapted to arid/semi-arid environments yet more sensitive to salt stress than bread wheat (BW). To enhance DW salinity tolerance, we resorted to chromosomally engineered materials with introgressions from allied halophytic Thinopyrum species. “Primary” recombinant lines (RLs), having portions of their 7AL arms distally replaced by 7el1L Th. ponticum segments, and “secondary” RLs, harboring Th. elongatum 7EL insertions “nested” into 7el1L segments, in addition to near-isogenic lines lacking any alien segment (CLs), cv. Om Rabia (OR) as salt tolerant control, and BW introgression lines with either most of 7el1 or the complete 7E chromosome substitution as additional CLs, were subjected to moderate (100 mM) and intense (200 mM) salt (NaCl) stress at early growth stages. The applied stress altered cell cycle progression, determining a general increase of cells in G1 and a reduction in S phase. Assessment of morpho-physiological and biochemical traits overall showed that the presence of Thinopyrum spp. segments was associated with considerably increased salinity tolerance versus its absence. For relative water content, Na+ accumulation and K+ retention in roots and leaves, oxidative stress indicators (malondialdehyde and hydrogen peroxide) and antioxidant enzyme activities, the observed differences between stressed and unstressed RLs versus CLs was of similar magnitude in “primary” and “secondary” types, suggesting that tolerance factors might reside in defined 7el1L shared portion(s). Nonetheless, the incremental contribution of 7EL segments emerged in various instances, greatly mitigating the effects of salt stress on root and leaf growth and on the quantity of photosynthetic pigments, boosting accumulation of compatible solutes and minimizing the decrease of a powerful antioxidant like ascorbate. The seemingly synergistic effect of 7el1L + 7EL segments/genes made “secondary” RLs able to often exceed cv. OR and equal or better perform than BW lines. Thus, transfer of a suite of genes from halophytic germplasm by use of fine chromosome engineering strategies may well be the way forward to enhance salinity tolerance of glycophytes, even the sensitive DW.

Sansevieria Thunb. species are traditionally known as succulent ornamental plants worldwide. They are also cultivated for medicinal, fodder, soil conservation and fiber uses, and for their capacity to reduce environmental pollution. Sansevieria sexual propagation is limited by the lack of viable seeds, and reproduction is largely made via vegetative propagation by suckers or cuttings. For these reasons, genetic improvement by conventional breeding is limited. To overcome this problem and to address the increasing demand from customers for novel Sansevieria varieties, many commercial companies regularly use in vitro propagation, as is the case in the breeding process of several ornamental plants. In this paper, for the first time, we report a procedure for in vitro somatic embryogenesis and plant regeneration starting from three flower explants for seven different Sansevieria genotypes. Regeneration was attempted using stigmas/styles, anther/filament, and ovary which were cultured on a Murashige and Skoog solidified medium under three different plant growth regulator combinations. A good regeneration rate was obtained with all genotypes used under all culture conditions tested from every explant type, with percentages ranging from 0 to 73.3%. "Genetic stability" assessment of regenerated plants in respect to their mother plants was verified through flow cytometry analysis showing a high degree of uniformity, with only S. parva exhibiting a different level of DNA fluorescence among in vitro regenerated plants. This is an interesting achievement in the aim to produce true-to-type plants and new variants with desirable characteristics, both of which are desired features in ornamentals improvement.

The strengthening effect of a mild temperature treatment on the antimicrobial efficacy of essential oils has been widely reported, often leading to an underestimation or a misinterpretation of the product’s microbial status. In the present study, both a traditional culture-based method and Flow Cytometry (FCM) were applied to monitor the individual or combined effect of Origanum vulgare essential oil (OEO) and mild heat treatment on the culturability and viability of Escherichia coli in a conventional culture medium and in a fruit juice challenge test. The results obtained in the culture medium showed bacterial inactivation with an increasing treatment temperature (55 °C, 60 °C, 65 °C), highlighting an overestimation of the dead population using the culture-based method; in fact, when the FCM method was applied, the prevalence of injured bacterial cells in a viable but non-culturable (VBNC) state was observed. When commercial fruit juice with a pH of 3.8 and buffered at pH 7.0 was inoculated with E. coli ATCC 25922, a bactericidal action of OEO and a higher efficiency of the mild heat at 65 °C for 5′ combined with OEO were found. Overall, the combination of mild heat and OEO treatment represents a promising antimicrobial alternative to improve the safety of fruit juice.

Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology.1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy)2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes.3, 4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of whole-genome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms.5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification6, 7 within organized tissues, such as roots.8, 9 The trigger to develop this virtual issue came from the publication, in 2017, of an article entitled "Guidelines for the use of flow cytometry and cell sorting in immunological studies" in the European Journal of Immunology.10 As noted in that article, one of the advantages of FCM/FCS systems is that they are relatively simple to implement, with some qualifications, which coupled with the development of user-friendly devices and software during the last 15 years led to increasing applications in other areas, such as plant sciences. However, it is also simple to implement and operate the instruments inappropriately. This calls for a comprehensive and collective summary of the best practices when applying FCM/FCS to plants, as was done for immunology. The first consideration addresses the problem that plants, particularly the vascular plants, in their commonly recognized and utilized forms, exist not as single cell suspensions (typical of immunology) but as complex three-dimensional tissues comprising cells of irregular shapes, different types and functions, that collectively cooperate to produce the final plant form. Optimal methods for producing suspensions of cells, subcellular organelles and other components appropriate for FCM/FCS from these plant tissues and organs, are therefore one of the challenges discussed in this virtual issue. We are fully aware of the mantra that "junk in equals junk out" and having samples of the highest quality prior to FCM/FCS is a critical concern we also addressed here. The second consideration relates to the vast variety of different plant species found globally, and the recognition of the consummate ability of plants to produce secondary metabolites/products, affecting DNA staining and resulting fluorescence. Again, methods for recognizing and handling the different challenges provided to FCM/FCS methods by the biochemistries of the source samples are required. The third consideration focuses on the problem of addressing the non-critical application of FCM/FCS methods developed for mammalian cell systems (typically hematopoietic) to plants without careful consideration of their appropriateness. As it will be detailed in this virtual issue, application of FCM/FCS methods to mammalian cell systems almost exclusively occurs in the context of analysis of samples that comprise a majority, often close to 100%, of single cells in suspension. For plants, particularly when using these instruments and methods for the analysis of organelles in tissue homogenates, the objects of interest comprise a very minor subpopulation of the total particles passing through the instrument. Concepts such as placing initial gates around populations defined by forward scatter (FS) versus side scatter (SS), as routinely used to define leukocytes or other mammalian cells in culture, are at best meaningless and at worst can seriously hamper proper use of the instruments to provide meaningful results. Again, plants are sources of many forms of autofluorescence; in vascular plants, chloroplasts are intensely fluorescent in the red due to the presence of chlorophyll. Phycoerythrin, a red protein-pigment complex from the light-harvesting phycobiliprotein family found in red algae and cryptophytes, is commercially employed as a fluorescent label for antibodies in cytometry. The presence of autofluorescence can restrict the wavelength bandwidths for fluorescence excitation and emission, and this can affect how best to set up FCM/FCS instruments. In order to define and enunciate best practices, we drew together a network of volunteer authors, experienced in the application of FCM/FCS to plants. We have attempted to make this network as comprehensive as possible, to allow recommendations spanning all relevant life-forms, from the simplest photosynthetic microbes, to the more complex lower and vascular plants, and encompassing also the fungi. In this endeavor, we gratefully acknowledge the support of Wiley and Attila Tarnok, EIC of Cytometry. As indicated for the Guidelines in Immunology article,9 we do wish to keep our recommendations updated. Therefore, please send us your critical comments, new ideas, practical suggestions regarding best practices, and new articles that could be useful for possible future versions of this virtual issue. To end, we would like to remember that this virtual issue reflects the vision and dream of the late Jan Suda. He has been an inspiration for all of us, and, most certainly, he left us too soon. We are sure that his legacy will persist, not only in his home country, the Czech Republic, but also across the world. We sincerely hope this virtual issue of Cytometry Part A provides an appropriate tribute. Describing how FCCS can be optimally applied to plants requires information in two general areas (a) concerning the samples being prepared and analyzed, in our case focusing on the relevant physical features of plants as organisms, and (b) concerning the instrumentation being used for this analysis, centering on sample requirements imposed by engineering design and implementation. Green plants (Viridiplantae) constitute a monophyletic clade within the tree of life and comprise oxygenic photosynthetic eukaryotes.11 The group encompasses green algae and land plants, and further splits into major clades: the Chlorophyta,12 comprising only algae, and Streptophyta formed by several algal groups (such as Zygnematophyceae and Charophyceae;,)13, 14 and the land plants (Embryophyta). Land plants further split into several groups: the possibly paraphyletic assemblage of three bryophyte lineages (Bryophyta—mosses, Marchantiophyta—liverworts, and Anthocerotophyta—hornworts) and three sequentially-splitting lineages of vascular plants: lycopods (Lycopodiophyta), ferns and horsetails (Monilophyta) and seed plants (Spermatophyta). The latter group further splits into gymnosperms (Gymnospermae; i.e., conifers, cycads, Ginkgo, and gnetophytes) and angiosperms (Angiospermae;).15-17 The current review is primarily but not exclusively focused on flow cytometric applications in flowering plants, as they represent the most diverse and economically important, and therefore best studied, group of green plants. However, we mention the other green plant lineages where necessary and we also include other organisms that are found in various parts of the Tree of Life (algae in the traditional sense, fungi) and that share certain features of body organization and life style with plants (such as complex tissues or photosynthesis), and have for a long time been a subject of Botany in the broadest sense. The life cycles of algal groups are highly variable and may comprise stages only with haplophasic (n) or diplophasic (2n) chromosome numbers, although in other species both stages are present but in separate generations.18 All land plants exhibit a characteristic life cycle which alternates between a haplophasic gametophyte and a diplophasic sporophyte. Still, the relative importance of each stage in the life cycle differs between groups: while the gametophyte stage dominates in bryophytes (and is usually the tissue that is analyzed by FCM), the sporophyte stage dominates in the vascular plant groups and is the main focus of flow cytometric investigations. Despite a significant reduction in the size of the gametophyte (comprising only up to 3–4 cells/nuclei in flowering plants), there are flow cytometric applications focused on either the independent gametophyte or the spores of ferns or on pollen grains of seed plants.19 Unlike vascular plants, fungal life cycles are mostly haplophasic, with a short (often single-celled) diplophasic stage, although most fungi (the Dikarya, i.e., the Ascomycota and the Basidiomycota) are dikaryotic (n + n) in part of their life cycles. The evolution of plant genomes is dynamic, particularly in angiosperms, encompassing a range of genomic processes including multiple rounds of whole genome duplication (polyploidization,20, 21 chromosomal rearrangements22, 23 and the turnover and evolution of repetitive DNA (including mobile elements and satellite DNA).24, 25 This is mirrored in the tremendous variation in nuclear genome sizes across green plants in general (c. 11,850-fold; 2) and flowering plants in particular (2,400-fold variation; 3,4). This has crucial implications for flow cytometric applications both with respect to technical issues (a series of internal standards of different genome size is required) and also as a study topic per se (e.g. what are the mechanisms driving genome size evolution?). Similarly, the relative content of AT versus GC base pairs is highly variable in green plants, although this variation does not strictly correlate with nuclear DNA-content (e.g., 26). While the algal groups are mostly unicellular, or comprise a rather simple multicellular thallus (e.g., Ulva, Cladophora, or Chara), land plants form complex tissues and organs. The sporophyte of vascular plants typically differentiates into roots, stems and leaves (note that the floral parts of flowering plants are derived from the leaves). Similar (yet haplophasic and thus non-homologous) structures are found in the gametophytes of bryophytes: rhizoids, cauloids, and phylloids. The specific morphology and anatomy of green plants, as distinct from other eukaryotes, naturally has multiple implications/challenges for flow cytometric analysis. Firstly, we encounter cells having thick cell walls that render flow cytometric analysis of individual cells impossible. Instead, isolated protoplast and, more commonly, nuclear suspensions are used for the analysis of plant tissues.26-28 Secondly, two types of endosymbiotic organelles, each with their own genomes, are present in most plant cells, mitochondria and plastids, and FCM applications have been designed to analyze those organelles.29-31 Lastly, plants present a wide array of chemical compounds, so-called secondary metabolites, conferring protection against factors both abiotic (e.g., UV-light, frost) and biotic (e.g., herbivores, parasites). Some of these chemical compounds (for example, tannins) directly co-interact with the DNA-binding stains used in FCM, and significantly affect the quality and reliability of such analyses.32 Flow cytometry and cytometric sorting systems are assembled from distinct engineering modules which collectively function to determine the optical properties of suspensions of biological particles, and selectively isolate these particles, or subsets thereof, for subsequent analysis and processing. The particles are typically constrained hydrodynamically within an aqueous stream to flow singly through regions of intense light, almost exclusively provided by lasers that are focused on the stream. On illumination, the particles absorb and scatter light and, if associated with fluorochromes, subsequently emit fluorescence. The intensities of the scattered and fluorescent light pulses coming from each particle are then measured. Key elements in these modules are (a) a flow cell, which spatially positions and aligns the flow stream containing the particles with the excitation light and detection axes, (b) light scatter and fluorescence detectors, screened by wavelength-appropriate filters and oriented orthogonally to the direction of the flow stream and the excitation light path, (c) electronic circuitry including analog-to-digital converters (ADCs) which convert the voltage pulses emerging from the detectors into digital values corresponding to the outputs from the individual particles, (d) computational architecture to process and store the information from these pulses for further analysis, or to use them immediately for processing sort-related decisions, and (e) mechanisms to implement individual, high-speed sorting of the particles, based on preselected combinations of optical characteristics. One of the first implementations of flow sorting, and one of the most influential, was described by Bonner et al.33 for characterization and isolation of various mammalian cell types including those of the hematopoietic system. To date, immunological applications represent the largest fraction of cytometric activities, worldwide. Most flow sorters employ a version of this original implementation, which involves precise conversion of the flow stream into a series of individual droplets, electromechanically synchronized to appear at a fixed distance below the point of laser interception (Figure 1). Based on the degree of sample dilution, some of these droplets contain the cells of interest, and can be selectively displaced into collection vessels by a process of charging the droplet at the point of its detachment from the flow stream followed by passage through a fixed electrostatic field. The rates of sorting depend on the size of the cells, which determines the size of the flow tip, and the rate of flow of the fluid stream.34 Advances in the area of instrument development have included multiplexed excitation and detection modalities to comprehensively cover the excitation and fluorescence emission spectra of the available fluorochromes.35 Recently, spectral analysis has been demonstrated as an alternative to conventional light filters in FCM.36, 37 Other advances include the use of flow tips that accommodate cells and biological particles that are larger, and sometimes much larger, than mammalian blood cells, drastic reductions in overall instrument sizes, footprints, and purchase costs, full replacement of analog by digital signal processing and the use of miniaturized fluidics systems with corresponding improvements in accuracy and reliability, and accompanied by reductions in costs of maintenance. Flow cytometry and flow cytometric sorting are not new methods. However, their use in Plant Biology has grown dramatically in the last decades, and in some cases, such as genome size measurements, these technologies have come to dominate. At the same time, instruments and associated protocols continue to be improved and expanded (e.g., bead beating, the use of tissues other than leaves, dry tissue). The literature now includes many resources outlining methods, theoretical issues, and limitations of methods (e.g., the "Flow Cytometry with Plant Cells" book,38 ESACP guidelines http://www.classimed.de/esacflow.html). However, despite this progress, it is clear from some recent publications that experimental design and manuscript review have not always kept pace with what we know about the application of FCM and cytometric sorting to plants, and this has adversely affected the quality of the results and the conclusions drawn. Contributing factors include: Examples of poor practices and erroneous theories developed as a consequence of these practices, identified by Jan Suda in the original draft, include flax genotrophs and problems with intraspecific variation reports, as reviewed in Greilhuber.35 Recent tendencies in manuscripts to justify the use of dry tissue based on existing literature frequently lack acknowledgment of necessary precautions from the prior literature. The main objective of this virtual issue is to outline key experimental issues and associated guidelines (under the heading of "Best Practices") that researchers are recommended to follow, and to provide the rationale for these recommendations, such that the guidelines may be modified with confidence as new applications emerge. We also identify those areas where the establishment of clear guidelines will require additional empirical data or theoretical work. Such guidelines will benefit researchers, facility managers, journal editors, and reviewers, since they should serve to guarantee high-quality results through elimination (or, at the very least, minimization) of artifactual variation from future research submitted for publication, as well as providing a means to identify artifacts within the published literature. The emphasis will be on providing guidelines for reviewers and for experimental design. This will NOT be a methods virtual issue in the sense of providing protocols: these are well-covered elsewhere (e.g.,27 "Flow Cytometry with Plant Cells" book,38 the supplemental material of Kron et al.,39 online resources). The authors have no conflicts of interest to declare. David Galbraith: Conceptualization; writing-original draft; writing-review and editing. João Loureiro: Conceptualization; writing-original draft; writing-review and editing. Ioanna Antoniadi: Writing-review and editing. Jillian Bainard: Writing-review and editing. Petr Bureš: Writing-review and editing. Petr Cápal: Writing-review and editing. Mariana Castro: Writing-review and editing. Sílvia Castro: Writing-review and editing. Martin Čertner: Writing-review and editing. Dora Čertnerová: Writing-review and editing. Zuzana Chumová: Writing-review and editing. Jaroslav Dolezel: Writing-review and editing. Debora Giorgi: Writing-review and editing. Brian Husband: Writing-review and editing. Filip Kolar: Writing-review and editing. Petr Koutecký: Writing-review and editing. Paul Kron: Writing-review and editing. Ilia Leitch: Writing-review and editing. Karin Ljung: Writing-review and editing. Sara Lopes: Writing-review and editing. Magdalena Lučanová: Writing-review and editing. Sergio Lucretti: Writing-review and editing. Wen Ma: Writing-review and editing. Susanne Melzer: Writing-review and editing. István Molnár: Writing-review and editing. Ondřej Novák: Writing-review and editing. Nicole Poulton: Writing-review and editing. Vladimír Skalický: Writing-review and editing. Elwira Sliwinska: Writing-review and editing. Petr Šmarda: Writing-review and editing. Tyler Smith: Writing-review and editing. Guiling Sun: Writing-review and editing. Pedro Talhinhas: Writing-review and editing. Attila Tárnok: Writing-review and editing. Eva Temsch: Writing-review and editing. Pavel Trávnícek: Writing-review and editing. Tomas Urfus: Writing-review and editing.

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line were crossed and backrossed four times. At each step electrophoretic selection for the seeds that possessed, simultaneously, the complete set of high molecular weight glutenin subunits of N11 and the two high molecular weight secalins of rye, present in the 2D(2R) line, was carried out. Molecular cytogenetic analyses of the BC4F8 generation revealed that the selection carried out produced a disomic addition line (2n = 44). The pair of additional chromosomes consisted of the long arm of chromosome 1R (1RL) from rye fused with the satellite body of the wheat chromosome 6B. Rheological analyses revealed that the dough obtained by the new addition line had higher quality characteristics when compared with the two parents. The role of the two additional high molecular weight secalins, present in the disomic addition line, in influencing improved dough characteristics is discussed.

Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.

Before the recent advances in molecular biology and sequencing techniques enabled a deep knowledge of the complete nucleotide sequence of many plant genomes, cytological and karyological studies have provided the first information concerning the genome organization of living beings. As stated by Figueroa and Bass (2010), plant cytogeneticists were among the earliest researchers who started to visualize genomes nearly a century before the first plant genome was sequenced (The Arabidopsis genome initiative 2000). In spite of the high development of next-generation sequencing approach and of the dramatic increase in available data regarding sequenced genomes, the cytological analysis of cell nucleus content remains a valuable tool for evolutionary studies and for structural and functional genomic research. Since Wilhelm Gottfried von Waldeyer-Hartz coined the term chromosome in 1888, many cytological techniques have been developed to disclose nucleus content and to allow a detailed description of the chromosome complement of plant species. Recently, some of these techniques have been applied to the cytological analysis and the molecular cytogenetic characterization of Cynara cardunculus complement, a traditional vegetable crop of the Mediterranean basin. The DNA content and a detailed karyotype of the two cultivated botanical varieties C. cardunculus var. altilis DC (cultivated cardoon) and C. cardunculus L. var. scolymus L. (globe artichoke) have been reported (Khaldi et al. 2014; Falistocco 2016; Giorgi et al. 2016). In this chapter, some of the used methodological approaches and the main results obtained by different authors will be discussed.

Orchids of the genus Dendrobium hold a high economical value in the international markets both as pot plants or cut flowers, and for the production of some metabolites with antioxidant and anti-tumoral activities. Manipulating ploidy levels of Dendrobium species is one of the possible methods to develop new varieties with increased ornamental value and a higher production of secondary metabolites. In this work, we present a new and fast flow cytometry approach to obtain and select Dendrobium phalaenopsis × Dendrobium loddigesii polyploids, through an early in vitro screening on protocorm like bodies (PLBs) after antimitotic treatment. Our approach allows the identification of the best time of treatment on control PLBs and the assessment of best conditions for polyploid recovery just one month after treatments, by using Cycle Value. We were able to discard about the two-third of the unchanged material by drastically reducing the explants to work with and the corresponding costs. Different conditions, regarding concentrations and exposition time, were tested using colchicine or amiprophos-methyl (APM). A high polyploids recovery, up to 80%, were obtained with both antimitotic agents, and those materials were further characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), to identify independent polyploids explants with increased levels in high-value molecules as shihunidine and hircinol, together with stochastic events and genotype-specific metabolite fluctuations.

The complexity of the contemporary world requires an in-depth rethinking of the role of design and designers. Its very multi-disciplinary and trans-disciplinary nature, its attempt to interpret the complexity and challenges of contemporary life, requires dealing with ethical issues and continually elaborating new tools which enable the strength deriving from its flexibility and ability to take cross fertilisations on board to be exploited to the full. The Mediterranean can, in particular, be an extremely interesting scenario for experimenting with new educational trajectories which can contribute to reconnecting and recreating that inter-personal and exchange nature which has always been its key characteristic, by means of cultural diversity promotion.

A number of leading crop species are either polyploid and/or hold a large genome as a result of the accumulation of highly repetitive sequences. These features hamper the assembly of complex genomes in spite of the fast development of new sequencing technologies (Next Generation Sequencing) and the availability of powerful bio-informatic tools. The chromosome approach, by enabling genome dissection into single chromosomes or chromosome arms via low sorting, can contribute to reduce genome complexity. In all plants, this approach is restricted to species, or special cytogenetic stocks, as in the case of wheat, containing chromosome types that differ in size from the standard complement. We have developed an overall robust and easy method called FISHIS (Fluorescence In Situ Hybridization in Suspension) which overcomes the constrain of chromosome size and DNA content differences, allowing chromosome low sorting on the base of luorescent hybridization labeling of chromosomes in suspension. FISHIS relies on readily available synthetic, luorescently labeled repetitive sequences and on a kaline DNA denaturation. We show that the method can discriminate between and hence isolate the A genome from the B genome of durum wheat (Triticum turgidum subsp. durum), a number of the chromosomes of the same species and of T. monococcum, as well as the whole complement of the diploid wheat relative Dasypyrum villosum (L.) Candargy.

Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus Linnaeus, 1753 var. scolymus Fiori, 1904) and leafy cardoon (C. cardunculus Linneaus, 1753 var. altilis De Candolle, 1838). Fluorescent In Situ Hybridization In Suspension (FISHIS) was applied to nuclei suspensions as a fast method for screening of labelling probes, before metaphase spread hybridization. Classic Fluorescent In Situ Hybridization (FISH) on slide, using repetitive telomeric and ribosomal sequences and Simple Sequence Repeats (SSRs) oligonucleotide as probes, identified homologous chromosome relationships and allowed development of molecular karyotypes for both varieties. The close phylogenetic relationship between globe artichoke and cardoon was supported by the very similar karyotypes but clear chromosomal structural variation was detected. In the light of the recent release of the globe artichoke genome sequencing, these results are relevant for future anchoring of the pseudomolecule sequence assemblies to specific chromosomes. In addition, the DNA content of the two crops has been determined by flow cytometry and a fast method for standard FISH on slide and methodological improvements for nuclei isolation are described.

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Artichoke is of great economic significance in the Mediterranean basin. Although Italy has the richest primary cultivated gene pool, only a limited number of varietal types are vegetatively propagated to fit market demand. This could lead to the erosion of genetic resources. The development of cryopreservation procedures for the ex situ conservation of the genetic resources of artichoke is advisable not only to counter genetic erosion, but also to develop a certified phytosanitary germplasm collection. In this study, several parameters were tested in order to set up a simple and efficient procedure for the cryopreservation of artichoke shoot tips, based on vitrification/one-step cooling, which was successfully applied to two spring cultivars ‘Grato 1’ and ‘Campagnano’. Cold acclimation prior to cooling was essential for tissue recovery and regrowth. The size of the shoot tips was also critical, and medium size tips (3–4 mm in length) proved to be the best responding starting material. The best survival percentage of cryopreserved shoot tips was 61% for ‘Grato1’ and 55% for ‘Campagnano’. Cryopreserved shoot tips developed directly into plantlets, which proved to be stable in terms of chromosome numbers. The cryopreservation procedure was notably effective in eliminating the endemic Artichoke Latent virus (ArLV) from both cultivars, irrespective of the shoot tip size used, which were up to 10 times the size used in meristem cultures for virus elimination. All plantlets analyzed for ArLV by RT-PCR were virus-free eight months after regeneration. An analysis of ArLV elimination and chromosome numbers of cryopreserved artichoke plants is presented here for the first time.

Cynara cardunculus L. (2n=34) is a traditional vegetable crop of the Mediterranean basin including two cultivated varieties: Cynara cardunculus L. var. scolymus L (globe artichoke), grown for its fleshy capitula, and C. cardunculus var. altilis DC (cardoon) grown for its succulent young leaves. Unlike other species belonging to the family of Asteraceae (i.e., sunflower, lettuce and chicory), the genome organization and the chromosome morphology both of globe artichoke and cardoon has not been sufficiently explored. To our knowledge, no karyotype analysis for this species is available so far. Different factors make very difficult the definition of chromosome morphology such as: the plant rigid cell wall, the presence of cellular debris, the thick cytoplasmic remains and the small size of the 34 chromosomes of Cynara cardunculus. In order to develop an effective chromosome preparation method for globe artichoke karyotyping here we evaluated different chromosome preparation systems and we observed the effect of some spindle inhibitors on globe artichoke cell cycle. The first Cynara cardunculus L var. scolymus L. karyotype is presented.