An advanced image analysis system for evaluation of somatic embryo development

Image analysis system for assessment of immunohistochemically stained proliferative marker (MIB-1) in oesophageal squamous cell carcinoma

The asexual, non-zygotic (somatic) embryogenesis was reported over 60 years ago. It is a unique process in which somatic cell transforms into embryo in cultured condition affirming cellular totipotency. Since its first report, somatic embryogenesis has been noted in different plant genera of a dicot, monocot, and lower fern groups. In the present chapter, an attempt has been made in describing somatic embryogenesis and its current status in periwinkle, Catharanthus roseus—an immensely important medicinal plant. The plant has anticancerous properties, the yield of alkaloids specific to this disease is however low and needs enrichment. The early embryogenic cells could serve as an important tissue for generating new cell lines with improved yield and related traits. Here, the influence of explants, the involvement of plant growth regulators, and the role of other regulating factors in establishing Catharanthus embryogenic tissues are discussed. The somatic embryo development, structure, progress, i.e., maturation, germination of embryo are also discussed with the help of Scanning Electron Microscopic method. The secondary somatic embryogenesis cycle, the embryo irregularities, and the role of protoplast culture in somatic hybridization is briefly deliberated taking Catharanthus as an example. Liquid overlaying at varying levels are described of which 0.50 ml overlaying on solid medium improved embryogenic growth and embryo number significantly. Successful artificial seed production, storage at low temperature, and plant regeneration are discussed by optimizing sodium alginate (2.5%) and calcium chloride (100 mM) solution. The proteomic approach has recently been exploited to investigate non-embryogenic, embryogenic tissues, embryo development, and other events at molecular level. In Catharanthus, over 1000 new proteins were identified from embryogenic callus, which was absent in non-embryogenic tissues. Some important proteins identified at various stages of embryogenesis are catalase, glutathione S transferase, superoxide dismutase, heat shock protein 70 (HSP70), HSP 90, and cytochrome C. Conservation of embryogenic tissues is important as it produces embryos and plantlets continuously. The cryopreservation protocol of Catharanthus is discussed by mentioning the role of osmotica, various cryoprotective agents, and the exposure of dehydration. Somatic embryo regenerated plants are true to type when developed directly on explant, but various types of variations are noted in plants developed from callus, suspension, and similar other differentiated tissues. In Catharanthus, the genome size or the 2C DNA of somatic embryo regenerated plant was noted to be 1.516 pg, which is similar to field grown plant DNA. No alteration in genome size was noted although the plants were produced through callusing. All this accumulated information on Catharanthus will be very valuable in practical and basic applications as it broadens the knowledge of cellular differentiation and dedifferentiation processes; at the same time, the methodology will augur fast multiplication of raw materials for the synthesis and enrichment of alkaloids.KeywordsCryopreservationGenome sizePlant growth regulatorsProteomicsProtoplastSignalingSomatic embryoSynthetic seed

Nutrient limitation and inhibitor accumulation have been shown to impede the development of somatic embryos of carrots in batch cultures. To improve the development of embryos, semicontinuous cultures with daily medium replenishment were performed. An image analysis system capable of identifying normal and abnormal embryos was used to facilitate the kinetic study. At a high daily medium replenishment rate of 20%, the biomass production was also higher, but not the embryo concentration. Total embryo and torpedo embryo concentrations were both higher at a 10% medium replenishment rate than at 5% and 20%. The profiles of embryo concentrations under three medium replenishment rates appeared similar until a late stage of cultivation; however, statistical analysis of the morphological features distribution revealed that significant differences were discernible earlier. For the formation of mature embryos, the optimal daily medium replenishment is judged to be in the range of 10–20%. In this study, more than 20000 embryos were classified and counted. The information on population kinetics and statistical comparison were made possible by the availability of our image analysis system.

Medical image computing has become one of the most challenging fields in medical informatics. In image-based diagnostics of the future software assistance will become more and more important, and image analysis systems integrating advanced image computing methods are needed to extract quantitative image parameters to characterize the state and changes of image structures of interest (e.g. tumors, organs, vessels, bones etc.) in a reproducible and objective way. Furthermore, in the field of software-assisted and navigated surgery medical image computing methods play a key role and have opened up new perspectives for patient treatment. However, further developments are needed to increase the grade of automation, accuracy, reproducibility and robustness. Moreover, the systems developed have to be integrated into the clinical workflow. For the development of advanced image computing systems methods of different scientific fields have to be adapted and used in combination. The principal methodologies in medical image computing are the following: image segmentation, image registration, image analysis for quantification and computer assisted image interpretation, modeling and simulation as well as visualization and virtual reality. Especially, model-based image computing techniques open up new perspectives for prediction of organ changes and risk analysis of patients and will gain importance in diagnostic and therapy of the future. From a methodical point of view the authors identify the following future trends and perspectives in medical image computing: development of optimized application-specific systems and integration into the clinical workflow, enhanced computational models for image analysis and virtual reality training systems, integration of different image computing methods, further integration of multimodal image data and biosignals and advanced methods for 4D medical image computing. The development of image analysis systems for diagnostic support or operation planning is a complex interdisciplinary process. Image computing methods enable new insights into the patient's image data and have the future potential to improve medical diagnostics and patient treatment.

Several coniferous species can today be propagated through somatic embryogenesis, but for species belonging to the Pinus genus, there are still problems related to the small number of genotypes from which embryogenic cultures can be established and the low yield of high-quality cotyledonary embryos. In order to pinpoint molecular processes that might be disturbed during somatic embryogenesis in Scots pine, we have analyzed the expression pattern of selected transcripts during development of somatic embryos in a normal and an abnormal cell line of Scots pine. The selected transcripts have been previously shown to be differentially expressed during early zygotic embryogenesis in Scots pine (Merino et al. 2016). Based on the transcripts that accumulated differentially between the two cell lines, we conclude that the apical–basal polarization in early somatic embryos and the transition from the morphogenic phase to the maturation phase are disturbed in the abnormal cell line. A comparison between transcript accumulation during somatic and zygotic embryogenesis highlighted problems with the continuous embryo degeneration processes in embryogenic cultures of Scots pine. Transcripts that were highly abundant during the cleavage process in zygotic embryos showed different accumulation patterns during somatic embryogenesis in Scots pine compared to those in Norway spruce. Furthermore, altering the expression of these transcripts in embryogenic cultures of Norway spruce resulted in a changed proliferation pattern of the embryos so that they became more similar to proliferating somatic embryos of Scots pine. Taken together, our results provide a deeper understanding of the deviations in abnormal cell lines and indicate that embryogenic cultures of Scots pine are proliferating by a cleavage-like process.

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.

We have developed and introduced a digital multi-modality image infrastructure in a multi-vender networked environment. This cardiology PACS (THOPACS) provides facilities for network communication between all DICOM-image generating systems in the Thoraxcentre. It facilitates hospital-wide access to all cardiology images and analysis results. THOPACS is integrated in the medical workstation of our hospital offering simultaneous presentation of images and related reports along with test results. This paper focuses mainly on the solutions for handling huge datasets, the archiving strategy, the automatic uploading of images from other hospitals, workflow support and the interoperability with image analysis systems.

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.

A research on the somatic embryogenesis in Lithospermum erythrorhizol1 has been conducted. Embryogenic callus was inoculated in EIM9 liquid medium (modification ofLS medium), i.e. L2PVP (initiation medium) and the development of somatic embryo was observed. Ten J.1M and 100).lM methyl jasmonate was added into L3PVP medium (differentiation medium) for somatic emb yo maLUration. The purposes of this research were to observe tlle development of somatic embryo and to observe the effect of methyl jasmonate on maturation of the somatic embryo in L erylhrorhizol/ . The results showed that somatic embryogenesis in L erythrorhizon derived from single cells differentiated further forming proembryo, globular. heart, torpedo and cotyledon stage. Treatment with 10 f.1M and 100).lM methyl jasmonate induced maturation of somatic embryos (cotyledon stage) after transferring them to embryo development medium (L4PVP).

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Light microscope analysis of cytological smears of suspicious lesions of the oral cavity is used as a method for detecting early cancer in the oral cavity. The sensitivity of this approach can be improved by quantitative analysis of the cells in the cytological smears. We have compared the efficiency of planimetry and the Vids V system of image analysis, as quantitative methods for discriminating between normal and abnormal cells in cytological smears of suspicious lesions in the oral cavity. Both methods detected an increase in nuclear area and a decrease in cytoplasmic area in abnormal epithelial cells from dysplastic lesions of increasing severity. However, image analysis was better able to discriminate between benign and malignant cells on the basis of nuclear size. Thus the Vids V system of image analysis is more appropriate than planimetry for quantitative analysis of cytological smears from the oral cavity.

Key message Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12–13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

An in-situ image acquisition and analysis system was developed to characterize the dynamics of particle size distribution (PSD) during flocculation. The system consisted of a flocculator coupled with an external flow-through cell for observation and photography, a microscopic charge-coupled device video recorder with backlighting, and an image analyzer. This non-intrusive side-stream setup was used to record the evolution of the PSD to determine the flocculation dynamics of two types of particle systems: Clean kaolin, kaolin coated with natural organic matter (NOM). The results were discussed and the error induced during measurement was evaluated.

Development of somatic embryos from coffee leaf explants has been studied in detail by scanning electron microscopy (SEM). Friable embryogenic callus is formed by single spherical cell aggregates of about 20μm diameter. They are the putative initial cells of somatic embryos or embryo mother cells. The fine structure of somatic embryos was clearly identified during the following sequential developmental stages : single spherical cell, early globular shape with conspicuous suspensor, typical globular shape, elongated shape, early heart shape, typical heart shape, torpedo shape, and finally cotyledonary stage embryo with well-developed suspensor. The high frequency of somatic embryos was found in callus tissues derived from mature leaves under a two-step culture method. Low frequency somatic embryogenesis was found on the leaf edges associated with very small callus formation. Low frequency somatic embryos were observed only in young leaves under the one-step culture method. In this SEM investigation, it is demonstrated that the sequential events leading to coffee somatic embryogenesis arise through one unicellular pathway derived from coffee somatic embryo mother cells. A schematic representation of the sequential stages of coffee somatic embryo development is provided.

Coffee is one of the most important crops cultivated in the world for use in beverages and confectionaries. Embryogenesis is a complex process that begins with a single cell and ends with the formation of mature embryos. Somatic embryo development involves accumulation of complex metabolites and storage reserves. This present experiment identified and quantified endogenous phytocomponents and amino acids present during somatic embryogenesis of ‘Ruiru 11’. Laboratory experiments for this study were set up in the Coffee Research Institute, Kenya at Ruiru. Third leaf pair explants were excised from 8-month-old greenhouse-grown mother plants sterilized and cultured in half strength Murashige and Skoog basal salts augmented with Thidiazuron. Once embryos had developed, the cultures were analysed for phytocomponents using GCMS and HPLC. The results showed that palmitoleic and stearic acids were highest (23.3 µg/g and 69.9 µg/g respectively) in brown embryogenic cultures. Cis 7,8 epoxy-2-methyl octadecane was highest (253 µg/g) in green embryogenic cultures. (Z)-3-Tetradecene was highest (25 µg/g) in brown non-embryogenic cultures. Z, Z-3,13- Octadecedien-1-ol and (Z)-7-Hexadecenal were highest (32.1 µg/g and 70.2 µg/g respectively) in green embryogenic cultures. Alanine content was highest (4.4 µg/g) in embryos of brown cultures. Amino acids, fatty acids and their derivatives are potential biomarkers for embryogenesis. Other phytocomponents should be identified and their role in coffee somatic embryogenesis determined. Further studies regarding the status of the phytocomponents identified in the present study, especially in particular stages of embryo development are needed to propose treatments to improve coffee somatic embryo development.