Towards automated phenotyping in plant tissue culture

Plant in vitro culture techniques comprise important fundamental methods of modern plant research, propagation and breeding. Innovative scientific approaches to further develop the cultivation process, therefore, have the potential of far-reaching impact on many different areas. In particular, automation can increase efficiency of in vitro propagation, a domain currently con-strained by intensive manual labor. Automated phenotyping of plant in vitro culture bears the potential to extend the evaluation of in vitro plants from manual destructive endpoint measurements to continuous and objective digital quantification of plant traits. Consequently, this can lead to a better understanding of crucial developmental processes and will help to clarify the emergence of physiological disorders of plant in vitro cultures. The aim of this dissertation was to investigate and exemplify the potential of optical sensing methods and machine learning in plant in vitro culture from an interdisciplinary point of view. A novel robotic phenotyping system for automated, non-destructive, multi-dimensional in situ detection of plant traits based on low-cost sensor technology was con-ceptualized, developed and tested. Various sensor technologies, including an RGB camera, a laser distance sensor, a micro spectrometer, and a thermal camera, were applied partly for the first time under these challenging conditions and evaluated with respect to the resulting data quality and feasibility. In addition to the development of new dynamic, semi-automated data processing pipelines, the automatic acquisition of multisensory data across an entire subculture passage of plant in vitro cultures was demonstrated. This allowed novel time series images of different developmental processes of plant in vitro cultures and the emergence of physiological disorders to be captured in situ for the first time. The digital determination of relevant parameters such as projected plant area, average canopy height, and maximum plant height, was demonstrated, which can be used as critical descriptors of plant growth performance in vitro. In addition, a novel method of non-destructive quantification of media volume by depth data was developed which may allow monitoring of water uptake by plants and evaporation from the culture medium. The phenotyping system was used to investigate the etiology of the physiological growth anomaly hyperhydricity. Therefore, digital monitoring of the morphology and along with spectro-scopic studies of reflectance behavior over time were conducted. The new optical characteristics identified by classical spectral analysis, such as reduced reflectance and major absorption peaks of hyperhydricity in the SWIR region could be validated to be the main discriminating features by a trained support vector machine with a balanced accuracy of 84% on test set, demonstrating the feasibility of a spectral detection of hyperhydricity. In addition, an RGB image dataset was used for automated detection of hyperhydricity using deep neural networks. The high-performance metrics with precision of 83.8% and recall of 95.7% on test images underscore the presence of for detection sufficient number of discriminating features within the spatial RGB data, thus a second approach is proposed for automatic detection of hyperhydricity based on RGB images. The resulting multimodal sensor data sets of the robotic phenotyping system were tested as a supporting tool of an e-learning module in higher education to increase the digital skills in the field of sensing, data processing and data analysis, and evaluated by means of a student survey. This proof-of-concept study revealed an overall high level of acceptance and advocacy by students with 70% good to very good rating. However, with increased complexity of the learning task, stu-dents experienced excessive demands and rated the respective session lower. In summary, this study is expected to pave the way for increased use of automated sensor-based phenotyping in conjunction with machine learning in plant research and commercial mi-cropropagation in the future.Die pflanzliche In-vitro-Kultur umfasst wichtige grundlegende Methoden der modernen Pflanzenforschung, -vermehrung und -züchtung. Innovative wissenschaftliche Ansätze zur Wei-terentwicklung des Kultivierungsprozess können daher weitreichenden Einfluss auf viele unter-schiedliche Bereiche haben. Insbesondere die Automatisierung kann die Effizienz der In-vitro-Vermehrung steigern, die derzeit durch die intensive manuelle Arbeit beschränkt wird. Automa-tisierte Phänotypisierung von In-vitro-Kulturen ermöglicht es, die Erfassung von manuellen de-struktiven Endpunktmessungen auf eine kontinuierliche, objektive und digitale Quantifizierung der Pflanzenmerkmale auszuweiten. Dies kann zu einem besseren Verständnis entscheidender Entwicklungsprozesse führen und die Entstehung physiologischer Störungen zu klären. Ziel dieser Dissertation war es, das Potential optischer Erfassungsmethoden und des maschinellen Lernens für die pflanzliche In-vitro-Kultur unter interdisziplinären Gesichtspunk-ten zu untersuchen und exemplarisch aufzuzeigen. Ein neuartiger Phänotypisierungsroboter zur automatisierten, zerstörungsfreien, mehrdimensionalen In-situ-Erfassung von Pflanzenmerkmalen wurde auf Basis kostengünstiger Sensortechnik entwickelt. Unterschiedliche Sensortechnologien, darunter eine RGB-Kamera, ein Laser-Distanzsensor, ein Mikrospektrometer und eine Wärmebildkamera, wurden teils zum ersten Mal unter diesen schwierigen Bedingungen eingesetzt und im Hinblick auf die resultierende Datenqualität und Realisierbarkeit bewertet. Neben der Entwicklung dynamischer, halbautomatischer Datenverarbeitungspipelines, wurde die automatische Erfassung multisensorischer Daten über eine gesamte Subkulturpassage der In-vitro-Kulturen demonstriert. Dadurch konnte erstmals Zeitrafferaufnahmen verschiedener Ent-wicklungsprozesse von pflanzlichen In-vitro-Kulturen und das Auftreten von physiologischen Störungen in situ erfasst werden. Die digitale Bestimmung relevanter Kenngrößen wie der proji-zierten Pflanzenfläche, der durchschnittlichen Bestandshöhe und der maximalen Pflanzenhöhe wurde demonstriert, die als wichtige Deskriptoren für das pflanzliche Wachstum dienen können. Darüber hinaus konnte eine neue Methode für die Pflanzenwissenschaften entwickelt werden, um die Wasseraufnahme von Pflanzen und die Verdunstung von Kulturmedien auf der Grundlage einer zerstörungsfreien Quantifizierung des Medienvolumens zu überwachen. Der Phänotypisierungsroboter wurde zur Untersuchung der Entstehung der Wachs-tumsanomalie Hyperhydrizität eingesetzt. Hierfür wurden ein digitales Monitoring der Morpho-logie der Explantate mit begleitenden spektroskopischen Untersuchungen des Reflexionsverhal-tens im Zeitverlauf durchgeführt. Die durch Spektralanalyse identifizierten optischen Merkmale, wie den reduzierter Reflexionsgrad und die Hauptabsorptionspeaks der Hyperhydrizität in der SWIR-Region, konnten als die wichtigsten Unterscheidungsmerkmale durch ein Support-Vektor-Maschine-Model mit einer Genauigkeit von 84% auf dem Testsatz validiert werden und damit Machbarkeit der spektrale Identifizierung von Hyperhydrizität aufzeigen. Darüber wurde für die automatische Detektion der Hyperhydrizität auf Basis von RGB-Bildern ein neuronales Netz trainiert. Die hohen Kennzahlen im Testdatensatz wie die Präzision von 83,8 % und einem Recall von 95,7 % unterstreichen das Vorhandensein einer für die Erkennung ausreichenden Anzahl von Unterscheidungsmerkmalen innerhalb der räumlichen RGB-Daten. Somit konnte ein zweiter An-satz der automatischen Detektion von Hyperhydrizität durch RGB-Bilder präsentiert werden. Die resultierenden Sensordatensätze des Phänotypisierungsroboters wurden als unter-stützendes Werkzeug eines E-Learning Moduls zur Steigerung digitaler Kompetenzen im Bereich Sensortechnik, Datenverarbeitung und -auswertung in der Hochschulausbildung erprobt und an-hand der Befragung von Studierenden evaluiert. Diese Machbarkeitsstudie ergab eine insgesamt hohe Akzeptanz durch die Studierenden mit 70% guten bis sehr guten Bewertungen. Mit zuneh-mender Komplexität der Lernaufgabe fühlten sich die Studierenden jedoch überfordert und bewerteten die jeweilige Session schlechter. Zusammenfassend zielt diese Arbeit darauf ab den Weg für einen verstärkten Einsatz der automatisierten, sensorbasierten Phänotypisierung in Kombination mit den Techniken des ma-schinellen Lernens der Forschung und der kommerziellen Mikrovermehrung zukünftig zu ebnen.Bundesministerium für Ernährung und Landwirtschaft (BMEL)/Digitale Experimentierfelder/28DE103F18/E

Low-cost and automated phenotyping system “Phenomenon” for multi-sensor in situ monitoring in plant in vitro culture

Background: The current development of sensor technologies towards ever more cost-effective and powerful systems is steadily increasing the application of low-cost sensors in different horticultural sectors. In plant in vitro culture, as a fundamental technique for plant breeding and plant propagation, the majority of evaluation methods to describe the performance of these cultures are based on destructive approaches, limiting data to unique endpoint measurements. Therefore, a non-destructive phenotyping system capable of automated, continuous and objective quantification of in vitro plant traits is desirable. Results: An automated low-cost multi-sensor system acquiring phenotypic data of plant in vitro cultures was developed and evaluated. Unique hardware and software components were selected to construct a xyz-scanning system with an adequate accuracy for consistent data acquisition. Relevant plant growth predictors, such as projected area of explants and average canopy height were determined employing multi-sensory imaging and various developmental processes could be monitored and documented. The validation of the RGB image segmentation pipeline using a random forest classifier revealed very strong correlation with manual pixel annotation. Depth imaging by a laser distance sensor of plant in vitro cultures enabled the description of the dynamic behavior of the average canopy height, the maximum plant height, but also the culture media height and volume. Projected plant area in depth data by RANSAC (random sample consensus) segmentation approach well matched the projected plant area by RGB image processing pipeline. In addition, a successful proof of concept for in situ spectral fluorescence monitoring was achieved and challenges of thermal imaging were documented. Potential use cases for the digital quantification of key performance parameters in research and commercial application are discussed. Conclusion: The technical realization of “Phenomenon” allows phenotyping of plant in vitro cultures under highly challenging conditions and enables multi-sensory monitoring through closed vessels, ensuring the aseptic status of the cultures. Automated sensor application in plant tissue culture promises great potential for a non-destructive growth analysis enhancing commercial propagation as well as enabling research with novel digital parameters recorded over time

Collagen VI-Related Myopathy Caused by Compound Heterozygous Mutations of COL6A3 in a Consanguineous Kurdish Family

Collagen VI-related myopathies are caused by mutations of COL6A1, COL6A2, and COL6A3 and present with a wide phenotypic spectrum ranging from severe Ulrich congenital muscular dystrophy to mild Bethlem myopathy. Here, we report a consanguineous Kurdish family with 3 siblings affected by autosomal-recessive Bethlem myopathy caused by compound heterozygous mutations of COL6A3. We found the previously described missense mutation c.7447A > G/p.(Lys2483Glu) and a novel large deletion encompassing the exon 1-39 of the COL6A3 gene. Apart from the classical clinical symptoms, all patients had keratoconus, which expands the phenotype of the collagen VI-related myopathies

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies

Comparison of matched sibling donors versus unrelated donors in allogeneic stem cell transplantation for primary refractory acute myeloid leukemia: a study on behalf of the Acute Leukemia Working Party of the EBMT

Background: Primary refractory acute myeloid leukemia (PRF-AML) is associated with a dismal prognosis. Allogeneic stem cell transplantation (HSCT) in active disease is an alternative therapeutic strategy. The increased availability of unrelated donors together with the significant reduction in transplant-related mortality in recent years have opened the possibility for transplantation to a larger number of patients with PRF-AML. Moreover, transplant from unrelated donors may be associated with stronger graft-mediated anti-leukemic effect in comparison to transplantations from HLA-matched sibling donor, which may be of importance in the setting of PRF-AML. Methods: The current study aimed to address the issue of HSCT for PRF-AML and to compare the outcomes of HSCT from matched sibling donors (n = 660) versus unrelated donors (n = 381), for patients with PRF-AML between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate. Results: HSCT provide patients with PRF-AML a 2-year leukemia-free survival and overall survival of about 25 and 30%, respectively. In multivariate analysis, two predictive factors, cytogenetics and time from diagnosis to transplant, were associated with lower leukemia-free survival, whereas Karnofsky performance status at transplant >= 90% was associated with better leukemia-free survival (LFS). Concerning relapse incidence, cytogenetics and time from diagnosis to transplant were associated with increased relapse. Reduced intensity conditioning regimen was the only factor associated with lower non-relapse mortality. Conclusions: HSCT was able to rescue about one quarter of the patients with PRF-AML. The donor type did not have any impact on PRF patients' outcomes. In contrast, time to transplant was a major prognostic factor for LFS. For patients with PRF-AML who do not have a matched sibling donor, HSCT from an unrelated donor is a suitable option, and therefore, initiation of an early search for allocating a suitable donor is indicated

Die chinesische Flote

Università degli Studi di Triest