This work examines the selective deposition of metallic and semiconductor materials onto DNA templates for the fabrication of nanodevices. DNA origami provides a simple and robust method for folding DNA into a variety of shapes and patterns and makes it possible to create the complex templates needed for nanodevices, such as nanoelectronic circuits, plasmonics, and nanosensors. Metallization of DNA origami templates is essential for the fabrication of such nanodevices. In addition, selective deposition of semiconductor materials onto the DNA template is of importance for making many nanodevices such as nanocircuits. Metallization of DNA origami presents several challenges beyond those associated with the metallization of other DNA templates such as λ-DNA. All of these challenges were addressed in this study. DNA origami templates were seeded with Ag and then plated with Au via electroless deposition. Selective continuous metal deposition was achieved, with an average metallized height as small as 32 nm. The structure of T-shaped DNA origami was also retained after metallization. Following the metallization of complete origami, site-specific metallization of branched DNA origami was also demonstrated. To achieve this, staple strands at select locations on origami were replaced with staple strands modified with binding sites at the end. These binding sites then attached to thiolated DNA coated Au nanoparticles through base pairing. The continuous Au nanowires formed at designated sites on DNA origami after Au plating had an average width of 33 nm, with the smallest ones ~20 nm wide. The continuity of nanowires was verified by conductivity tests- the only tests of this nature of which I am aware. Moreover, predesigned sites on "circuit-shaped" DNA origami were successfully metallized. The selective deposition of a variety of materials onto DNA templates for the formation of continuous DNA-templated nanowires was also demonstrated. Specifically, an electroless Ni plating solution was developed to enable the fabrication of uniform and continuous DNA-templated Ni nanowires. Tests showed that these DNA-templated Ni nanowires were conductive. Moreover, continuous DNA-templated Bi2Te3 and/or Te nanowires have been fabricated through galvanic displacement of DNA-templated Ni and Cu nanowires. Altogether, these results represent important progress toward the realization of DNA-templated nanofabrication.

Tyrosine site-specific recombinases (Y-SSRs) are DNA editing enzymes that play a valuable role for the manipulation of genomes, due to their precision and versatility. They have been widely used in biotechnology and molecular biology for various applications, and are slowly finding their spot in gene therapy in recent years. However, the limited number of available Y-SSR systems and their often narrow target specificity have hindered the full potential of these enzymes for advanced genome engineering. In this PhD thesis, I conducted a comprehensive investigation of novel Y-SSRs and their potential for advancing genome engineering. This PhD thesis aims to address the current limitations in the genetic toolbox by identifying and characterizing novel Cre-type recombinases and demonstrating their impact on the directed evolution of designer recombinases for precise genome surgery. To achieve these aims, I developed in a collaboration a comprehensive prediction pipeline, combining a rational bioinformatical approach with knowledge of the biological functions of recombinases, to enable high success rate and high-throughput identification of novel tyrosine site-specific recombinase (Y-SSR) systems. Eight putative candidates were molecularly characterized in-depth to ensure their successful integration into future genome engineering applications. I assessed their activity in prokaryotes (E. coli) and eukaryotes (human cell lines), and determined their specificity in the sequence space of all known Cre- type target sites. The potential cytotoxicity associated with cryptic genomic recombination sites was also explored in the context of recombinase applicability. This approach allowed the identification of novel Y-SSRs with distinct target sites, enabling simultaneous use of multiple Y-SSR systems, and provided knowledge that will facilitate the assignment of novel and known recombinases to specific uses or organisms, ensuring their safe and effective implementation. The introduction of these novel Y-SSRs into the genome engineering toolbox opens up new possibilities for precise genome manipulation in various applications. The broader targetability offered by these enzymes could accelerate the development of novel gene therapies, as well as advance the understanding of gene function and regulation. Moreover, these recombinases could be used to design custom genetic circuits for synthetic biology, allowing researchers to create more complex and sophisticated cellular systems. Finally, I introduced the novel Y-SSRs into efforts aimed at developing designer recombinases for precise genome surgery, demonstrating their impact on accelerating the directed evolution process. Therapeutically relevant recombinases with altered DNA specificity have been developed for excision or inversion of specific DNA sequences. However, the potential for evolving recombinases capable of integrating large DNA cargos into naturally occurring lox-like sites in the human genome remained untapped so far. Thus, I embarked on evolving the Vika recombinase to mediate the integration of DNA cargo into a native human sequence. I discovered that Vika could integrate DNA into the voxH9 site in the human genome, and then, I enhanced the process through directed evolution. The evolved variants of Vika displayed a marked improvement in integration efficiency in bacterial systems. However, the translation of these results into mammalian systems has not yet been entirely successful. Despite this, the study laid the groundwork for future research to optimize the efficiency and applicability of Y-SSRs for genomic integration. In summary, this thesis made significant strides in the identification, characterization, and development of novel Y-SSRs for advanced genome engineering. The comprehensive prediction pipeline, combined with in-depth molecular characterization, has expanded the genetic toolbox to meet the growing demand for better genome editing tools. By exploring efficiency, cross-specificity, and potential cytotoxicity, this research lays the foundation for the safe and effective application of novel Y-SSRs in various therapeutic settings. Furthermore, by demonstrating the potential of these recombinases to improve efforts in creating designer recombinases through directed evolution, this research has opened new avenues for precise genome surgery. The successful development and implementation of these novel recombinases have the potential to revolutionize gene therapy, synthetic biology, and our understanding of gene function and regulation.

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Synthetic biology has ushered in a new age of molecular biology with the aim towards practical developments in disciplines ranging from medicine, agriculture, and industry. Presently, it remains difficult to manipulate the genomes of many organisms due to lack of genetic tools. These problems can be circumvented by cloning large fragments of DNA into strains where many genetic tools are in place, such as Saccharomyces cerevisiae. However, this organism is unable to directly transfer cloned DNA to other organisms and is unable to stably maintain DNA with a G+C content >40%. Many organisms relevant in biotechnology often have G+C content DNA >60%, and therefore are difficult to engineer. Here, the soil bacteria Sinorhizobium meliloti was chosen as a host strain to clone and manipulate large fragments of high G+C content DNA. S. meliloti is a Gram-negativeα-proteobacteria that forms symbiotic relationships with legumes to fix nitrogen. It has a multi-partite genome with a G+C content of 62.7% that includes a chromosome (3.65 Mb), the pSymA (1.35 Mb), and pSymB (1.68 Mb) replicons. A restriction endonuclease hsdR mutant strain lacking pSymA and pSymB was created and used in this study. Multi-host shuttle (MHS) vectors were constructed that allow for direct transfer and maintenance of DNA in E. coli, S. cerevisiae, and P. tricornutum. Characterization of strains was conducted to determine transduction, conjugation, and transformation frequencies, as well as stability of MHS plasmids. Furthermore, a proof-of-concept experiment was conducted to clone large plasmids (70-205 kb) with G+C content >58% via site-specific recombination at a landing pad in the MHS vector, which was then verified using colony PCR. This work demonstrates the usefulness of S. meliloti containing a MHS vector for cloning of large fragments with high G+C content DNA, a technology that may be used for several applications in both applied and basic research. / Thesis / Master of Science (MSc) / Synthetic biology is an emerging field that incorporates principles of molecular biology and engineering for the design and construction of biological systems for application in medicine, agriculture, and industry. Presently, it remains difficult to modify genomes of several organisms due to lack of available techniques. Yeast is currently used for the modification of large DNA pieces, however it is unable to transfer and maintain modified DNA with high G+C content. Here, the bacteria Sinorhizobium meliloti was used as a host organism to conduct genetic engineering due to its ability to maintain large DNA pieces with a high G+C content. Characterization experiments were conducted to assess the efficiency of this organism for this task. Using this strain, a proof-of-concept experiment to demonstrate the uptake and maintenance of large, high G+C DNA pieces was completed. This technology may be useful in biotechnology applications for engineering of large DNA pieces from industrially relevant organisms.

Iron deficiency chlorosis (IDC) is a frequent problem throughout many areas of the United States where soils are high in calcium carbonate (CaCO3), including the Blackland Prairie regions of Mississippi. The main objectives of this study were to 1) determine the effects of seven different cropping systems on IDC visual symptomology and grain yield in rainfed soybeans grown in calcareous soils, and 2) evaluate the effects of soil water tension (SWT) on IDC. Rotating soybeans with corn produced significantly higher yields for both tolerant and susceptible soybean varieties. IDC symptomology was worse, and yields were lower in cropping systems with lower average SWT, or wetter soils, throughout the growing season. Last, this study evaluated the use of multispectral imagery and apparent soil electrical conductivity (ECa) to identify IDC-prone areas of a field for the site-specific implementation of management strategies that produced higher yields in the plot-scale study.

Tesis por compendio / [ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31. / [CA] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31. / [EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch. / Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055 / Compendio

The two fields of acousmatic music and site-specific conceptual art take strikingly different approaches to the notions of space and place. In this document, I describe how these two areas of aesthetic research diverge and relate to each other, focusing on how their unique approaches can be implemented in the practice of site-specific music composition. The first part of this document surveys the distinctive features of each of these fields, describing the particular differences between them in their approach to space and place. The contradictions between the two approaches are then briefly analyzed in reference to Georgina Born's understanding of music as fundamentally multitextual. In the second part of the document, I describe in detail how I implemented a site-specific approach when composing "…threaded through," a 16-channel audio, 6 video, site-specific installation for the UNT College of Music Main Building. In this, I describe how both the space and place of the UNT College of Music Main Building influenced my musical choices, visual content, and approach to audio and visual spatialization. The final part of the document contains a detailed score for realizing "…threaded through" in the location of the UNT College of Music Main Building.

Der maschinengestützte Pflanzenmassesensor "Pendulum-Meter" kann online die teilflächenspezifischen Differenzierung der Bestandesfrischmassen und -trockenmassen der meisten Wachstumsstadien von Winterweizen, Winterroggen und Naßreis bestimmen. Das Pendulum-Meter ist in Weizen und Roggen sehr gut geeignet für die Stadien BBCH 39 bis 69, und mit geringerer Genauigkeit auch für die Stadien BBCH 32 bis 34. In Naßreis wurde die Biomasse in allen getesteten Wachstumsstadien von BBCH 25 bis BBCH 65 gut erfaßt. In früheren Wachstumsstadien ist eine Messung nicht möglich. Die Kraft-Winkel-Beziehung des Sensors ist nicht linear. Der wichtigste Faktor für diese Kontaktmessung ist der Biegewiderstand der Getreidehalme. Zur Reduzierung der Rohdaten zu repräsentativen Werten für die Parzellen sind sowohl der Mittelwert des Auslenkungswinkels und der Mittelwert des Vektors, als auch der Median des Auslenkungswinkels geeignet. Die Ergebnisse der Optimierungsversuche in bezug auf die Wiederholgenauigkeit der Meßwerte und die Abbildungsgenauigkeit der Pflanzenmasse zeigten eine große Bandbreite von geeigneten Einstellungsparametern und keine einzelnen optimalen Parameter. Trotzdem sollte die Pendelmasse mP niedrig sein, um eine Zerstörung einiger Pflanzen zu vermeiden. Die Höhe des Zylinderkörpers hA0 sollte die Halme berühren, um den Biegekontakt sicherzustellen. Und die Drehpunkthöhe hP sollte in Bestandeshöhe sein, um eine größtmögliche Bandbreite an Auslenkwinkeln für eine gegebene Biomasse zu erhalten. Die optimalen Einstellungen für hP, hA0, und mP sind in jedem Wachstumsstadium anders, aber eine einzige Pendeleinstellung für alle Wachstumsstadien ist möglich, jedoch läßt sie in vielen Wachstumsstadien eine gute Genauigkeit der Biomassebestimmung vermissen. Ohne Kalibrierung ermittelt das "Pendelum-Meter" immer noch gut die relative Verteilung der teilflächenspezifischen Biomasse, also der Heterogenität des Feldes. Wenn nur die Bestandesheterogenität von Interesse ist, können alle Pendeleinstellungen ohne Kalibrierung benutzt werden. Die Meßwiederholungen mit derselben Parametereinstellung zeigte eine Standardabweichung der Parzellenmittelwerte von weniger als 1°, in den meisten Fällen geringer als 0.3° für die meisten Wachstumsstadien. Der Variationskoeffizient ist für die meisten Einstellungsparameter geringer als 5 %, oft kleiner als 2 %, und er ist größer je kleiner die Bandbreite des gemessenen Winkels ist. Die Genauigkeit der Pflanzenmassebestimmung durch das Pendulum-Meter ist durch Bestimmtheitsmaße von 0.90 oder höher gekennzeichnet. Die lineare Abbildung zeigte dabei geringfügig niedrigere R2 als die quadratische, außer in Roggen, wo in den späteren Wachstumsstadien die Pflanzenmasse wesentlich besser quadratisch darstellt wurde. Auch zeigten die geplotteten Residuen dieser Regression allein im Roggen eine Verfälschung durch einen quadratischen Einfluß. Die Standardfehler dieser Regressionen zur Abbildung der Biomasse waren in der Regel geringer als 2 in Naßreis, geringer als 3 in Winterweizen, und geringer als 4 in Winterroggen, und mit dem Bestandeswachstum zunehmend. Die multiple und einfache Regression der Abhängigkeit der Meßwerte von den Parametereinstellungen des Sensors wurde stark von der Pflanzenmasse der Parzellen beeinflußt, weshalb eine Kalibrierung notwendig ist, wenn die Einstellungsparameter geändert werden. Die Geländeneigung lenkt das Pendel aus, ohne einen Getreidebestand zu messen, und benötigt eine Korrektur durch einen Neigungssensor. Die Fahrgeschwindigkeit muß konstant gehalten werden, da der gemessene Auslenkwinkel eine starke Abhängigkeit von der Geschwindigkeit zeigt, aber gleichzeitig auch von der Menge der Pflanzenmasse. Der Biomassesensor kann die Bestandesdichte bestimmen, wenn die Bestandeshöhe homogen ist, und bestimmt die Bestandeshöhe, wenn die Bestandesdichte konstant ist. Wind mit niedriger Geschwindigkeit ist verfälscht die Biomassemeßwerte nicht meßbar, aber bei höheren Windgeschwindigkeiten wird der Fehler größer. Weitere verfälschende Faktoren sind Unkräuter, das tägliche Pflanzenwachstum, die Neigung des Halmes, Vertiefungen der Fahrgasse, verschiedene Sorten, die eine Eichung des Biomassesensors unter den meisten Umständen notwendig machen. Die Genauigkeit des Meßprinzips wurde mit einem Geräteträger ermittelt, der seine Ausrichtung gegenüber dem Erdboden nicht veränderte, aber ein Traktor kann die Messungen durch seine Eigenbewegung verfälschen. Die Messungen des Biomassesensors können als Entscheidungsbasis zur teilflächenspezifischen Applikation von Wachstumsregeln und Fungiziden dienen, wenn auch die Entscheidungskriterien noch erarbeitet werden müssen. Teilflächenspezifische Applikationen von Wachstumsreglern und Fungiziden nach den Messungen des Pendulum-Meters, also nach der Pflanzenmasse, sind erfolgreich durchgeführt worden. Die Messungen des Pendulum-Meters können dabei als Entscheidungsbasis für die teilflächenspezifische Applikation von Wachstumsreglern und Fungiziden benutzt werden, obwohl die Beziehung zwischen Lager und Pflanzenmasse, und zwischen den meisten Schadpilzen und der Biomasse weitere Untersuchungen erfordert, genauso wie die Ermittlung der notwendigen Aufwandmenge an Wachstumsreglern und Fungiziden je nach Pflanzenmasse. Der Biomassesensor Pendulum-Meter ist für Kontrolle der mittleren und späten Applikationen von Wachstumsreglern und Fungiziden geeignet, hier die Stadien BBCH 32 - 59, aber nicht für die frühen Applikationen, hier die Stadien BBCH 25 - 31, wegen der fehlenden Eignung den Sensor in niedrigem Pflanzenbestand zu benutzen. In der Fungizidapplikation kann der Sensor ähnlich den LAI-Meßgeräten benutzt werden, und in der Ausbringung von Wachstumsreglern hat der Sensor eine große Gemeinsamkeit mit dem Widerstand gegen das Lagern. / The machine-based biomass sensor pendulum-meter can determine on-line the site-specific differentiation of cereal crop fresh masses and dry masses for the most growth-stages in winter wheat, winter rye, and irrigated rice. The pendulum-meter is well suited in wheat and rye for the growth-stages BBCH 39 to BBCH 69, and to a lesser degree for BBCH 34 and 32. Irrigated rice crop biomass was well sensed by the pendulum-meter at all tested growth-stages BBCH 25 to BBCH 65. Earlier growth-stages were not possible to measure. The angle-force relation of the pendulum-meter is non-linear. The most important factor for this contact measurement was found in the bending moment of resistance of the stems. For the reduction of the original data to representative plot values, the average of the angle, the average of the vector, and the median of the angle were suitable. The results of the optimisation trials, in terms of repeatability of the measurement and the accuracy of biomass determination, showed a wide range of suitable parameter settings and not a single optimal parameter. Nevertheless, the mass of the pendulum mP should be low to avoid destruction of the plants, the height of the cylindrical body hA0 should touch the stems to ensure bending contact, and the height of the pivot point hP should be at crop height to get a wide range of angles of deviation for a given range of biomass. For every growth-stage, the optimum hP, hA0, and mP are different, and although a single pendulum-meter setting for all growth-stages is possible, but lacking good accuracy of biomass determination in many growth-stages. Without calibration the pendulum-meter still senses well the relative distribution of the site-specific biomass, hence the field heterogeneity. When only the crop's heterogeneity is of interest, all pendulum settings can be used without calibration. The replicates with the same pendulum parameter settings show a standard deviation of the plot average of less than 1°, mostly less than 0.3° for the most growth-stages. The coefficient of variation is mostly less than 5 %, often less than 2 %, and it is higher the smaller the range of measured angles is. The accuracy of biomass determination of the pendulum-meter showed mostly R2 of 0.90 or higher. The linear regression performed with slightly lower R2 than the square, except for rye, where the square regression was much better for the late growth-stages. Solely in rye the plotted residuals showed a square bias. The standard errors of the regressions were less than 2 in rice, less than 3 in wheat, and less than 4 in rye, increasing during crop growth. The multiple and simple regression for the dependency of the measured angle on the parameter settings of the pendulum was strongly influenced by the biomass thus causing a re-calibration when changing the pendulum parameters. The slope of the terrain is deviating the pendulum-meter without crop and needed a correction by a slope sensor. The carrier speed has to be constant, and the angle of deviation is highly dependent on the carrier speed, but simultaneously dependent on the amount of biomass. The biomass sensor senses the tiller density, when the plant height is homogeneous, and the plant height when the tiller density is constant. Wind at low speeds is not a biasing factor, but at high wind speeds the bias will increase. Further biasing factors can be weeds, daily plant growth, stem inclination, tramline depth, and variety, thus requiring a re-calibration of the biomass sensor under most conditions. The accuracy of the measurement principle was determined with a carrier that was not changing its orientation towards the soil surface, but a tractor might bias the measurements because of its own movement. Site-specific application of growth-regulators and fungicides according to the measurements of the pendulum-meter, and hence the biomass, has been successfully conducted. The measurements of the pendulum-meter can be used as a decision base for the site-specific application of growth-regulators and fungicides, although the relationship between lodging and biomass, and between most fungi and biomass needs further examination, as well as determining the necessary amount of growth-regulators and fungicides according to biomass. The biomass sensor pendulum-meter is suitable for controlling the intermediate and late applications of growth-regulators and fungicides, here BBCH 32 - 59, but not for the early applications, here BBCH 25 - 31, due to impossibility of using the sensor in low plants. In fungicide application, the sensor can be used similar to LAI, and in growth-regulators sprayings, the sensor has in principle a high similarity with lodging resistance.