Transgenic Plants: Gene Constructs, Vector and Transformation Method

The human population has reached 7 billion by 2015 and is estimated to exceed 10 billion by the end of 2050. As such, crops which are the main food source must be produced at a higher pace in order to cater in tandem with the food demand. In the past, traditional plant breeders practice classical breeding techniques to propagate plants with desirable traits. However, traditional breeding technique lies in that only individuals of the same or closely related species can be crossbred. Moreover, traditional breeders will not be able to obtain traits which are not inherent within the gene pool of their target plants through classical breeding. With recent advancements in the field of genetic engineering, it is now possible to insert beneficial genes from a completely different species or even kingdom into a target plant, yielding transgenic plants with multiple ideal traits. To develop a transgenic plant, parameters such as vector constructions, transformation methods, transgene integration, and inheritance of transgene need to be carefully considered to ensure the success of the transformation event. Hence, this chapter aimed to provide an overview of transgenic plants’ development, its advantages and disadvantages, as well as its application for the betterment of mankind

Construction Of User-Friendly Plant Expression Vectors Using Rice Promoters

This project (3 months duration) was embedded within our ongoing projects on “Rice Functional Genomics”. A PhD student, Andrew Eamens was employed in this project to continue work on the development of user friendly plant expression vectors based on rice promoters. This work was started towards the end of Andrew’s PhD studentship. Using reporter genes containing a minimal promoter (enhancer trap) or intron splice acceptors (gene trap) in T-DNA or transposon tagging systems, several promoter sequences were identified by Andrew during his doctoral research and were used to produce plant expression vectors with tissue specific expression. The previously developed double right boarder (DRB) vector technology was used to construct a small group of user-friendly plant expression vectors with tissue-specific expression promoters. A new base binary vector construct (PDRB12dn) was constructed during this project period. The binary vector contained a promoterless reporter gene (sgfpS65T) mounted between the second right border (RB2) and the T-DNA left border (LB). The reporter gene is flanked upstream by a multiple cloning site (MCS) containing several unique restriction enzyme (RE) cleavage sites for easy cloning of putative promoter fragments. A total of 12 promoter fragments were also amplified by the polymerase chain reaction (PCR), ready for addition to the base vector. Cloning of individual promoter fragments is now in progress. The plant expression constructs being produced will enable the production of selectable marker free transgenic plants expressing GOIs in specific cells, tissues or organs

COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS

Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains

Method of introducing a plurality of genes into plants

A method of concurrently introducing multiple genes to plants and trees is provided. The method includes an Agrobacterium-mediated gene delivery system by which multiple genes together with a single selectable marker gene are simultaneously transferred and inserted into the genome of plants, including trees.https://digitalcommons.mtu.edu/patents/1034/thumbnail.jp

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Establishing a foundation for large DNA transfer to artificial minichromosomes and B insert platforms in maize

In plants, conventional genetic engineering methods limit the number of available traits that could potentially improve the quality of agriculture. Agrobacterium-mediated transformation and biolistic bombardment are tools used in transferring genes into plant cells, both of which result in random integrations into host genomes. The absence of targeting machinery, together with low DNA carrying capacity on most plasmid vectors, limit researchers to a few genes in a single modification experiment, a process that takes [about]1 year in most plant species. While stacking traits from independent genetic modifications allow for an increase in the number of transgenes in a single plant, recovery of all genes in subsequent generations becomes increasingly difficult due to independent segregation in meiosis. Alternatively, the use of binary bacterial artificial chromosomes (BiBACs), large insert cloning vectors, can maintain and transfer up to 300 kps, but are also subject to random integrations. Therefore, establishment of a BiBAC targeting system would be advantageous for researchers focusing on creating plant lines that contain several genes that work together to express complex traits, such as disease resistance clusters or whole biosynthetic pathways. Additionally, BiBAC targeting to a location outside the native chromosomal sets, such as an artificial minichromosome or B chromosome platform, would enable researchers to stack traits without disrupting endogenous sequences.Includes bibliographical reference

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

[EN] Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, readyto-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciensmediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA,P7760,Pef1¿, and PafpB constitutive promoters, and PglaA,PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.This work was supported by PROMETEO/2018/066 from "Conselleria d'Educacio" (Generalitat Valenciana, Comunitat Valenciana, Spain), grant PID2021-125858OB-100, and the Severo Ochoa Excellence Program CEX 2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe." EM-G was the recipient of a predoctoral grant FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future." SG holds a Juan de la Cierva Incorporacion grant (IJC 2020-042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR.Moreno-Giménez, E.; Gandía, M.; Sáez, Z.; Manzanares, P.; Yenush, L.; Orzáez Calatayud, DV.; Marcos, JF.... (2023). FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi. Frontiers in Bioengineering and Biotechnology. 11:1-17. https://doi.org/10.3389/fbioe.2023.12228121171

Less is more: strategies to remove marker genes from transgenic plants

Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification

DESIGN OF GENETIC ELEMENTS AND SOFTWARE TOOLS FOR PLANT SYNTHETIC BIOLOGY

Tesis por compendio[EN] Synthetic Biology is an emerging interdisciplinary field that aims to apply the engineering principles of modularity, abstraction and standardization to genetic engineering. The nascent branch of Synthetic Biology devoted to plants, Plant Synthetic Biology (PSB), offers new breeding possibilities for crops, potentially leading to enhanced resistance, higher yield, or increased nutritional quality. To this end, the molecular tools in the PSB toolbox need to be adapted accordingly, to become modular, standardized and more precise. Thus, the overall objective of this Thesis was to adapt, expand and refine DNA assembly tools for PSB to enable the incorporation of functional specifications to the description of standard genetic elements (phytobricks) and to facilitate the construction of increasingly complex and precise multigenic devices, including genome editing tools. The starting point of this Thesis was the modular DNA assembly method known as GoldenBraid (GB), based on type IIS restriction enzymes. To further optimize the GB construct-making process and to better catalog the phytobricks collection, a database and a set of software-tools were developed as described in Chapter 1. The final webbased software package, released as GB2.0, was made publicly available at www.gbcloning.upv.es. A detailed description of the functioning of GB2.0, exemplified with the building of a multigene construct for anthocyanin overproduction was also provided in Chapter 1. As the number and complexity of GB constructs increased, the next step forward consisted in the refinement of the standards with the incorporation of experimental information associated to each genetic element (described in Chapter 2). To this end, the GB package was reshaped into an improved version (GB3.0), which is a self-contained, fully traceable assembly system where the experimental data describing the functionality of each DNA element is displayed in the form of a standard datasheet. The utility of the technical specifications to anticipate the behavior of composite devices was exemplified with the combination of a chemical switch with a prototype of an anthocyanin overproduction module equivalent to the one described in Chapter 1, resulting in a dexamethasone-responsive anthocyanin device. Furthermore, Chapter 3 describes the adaptation and functional characterization of CRISPR/Cas9 genome engineering tools to the GB technology. The performance of the adapted tools for gene editing, transcriptional activation and repression was successfully validated by transient expression in N. benthamiana. Finally, Chapter 4 presents a practical implementation of GB technology for precision plant breeding. An intragenic construct comprising an intragenic selectable marker and a master regulator of the flavonoid biosynthesis was stably transformed in tomato resulting in fruits enhanced in flavonol content. All together, this Thesis shows the implementation of increasingly complex and precise genetic designs in plants using standard elements and modular tools following the principles of Synthetic Biology.[ES] La Biología Sintética es un campo emergente de carácter interdisciplinar que se fundamenta en la aplicación de los principios ingenieriles de modularidad, abstracción y estandarización a la ingeniería genética. Una nueva vertiente de la Biología Sintética aplicada a las plantas, la Biología Sintética Vegetal (BSV), ofrece nuevas posibilidades de mejora de cultivos que podrían llevar a una mejora de la resistencia, a una mayor productividad, o a un aumento de la calidad nutricional. Sin embargo, para alcanzar este fin las herramientas moleculares disponibles en estos momentos para BSV deben ser adaptadas para convertirse en modulares, estándares y más precisas. Por ello se planteó como objetivo general de esta Tesis adaptar, expandir y refinar las herramientas de ensamblaje de DNA de la BSV para permitir la incorporación de especificaciones funcionales en la descripción de elementos genéticos estándar (fitobricks) y facilitar la construcción de estructuras multigénicas cada vez más complejas y precisas, incluyendo herramientas de editado genético. El punto de partida de esta Tesis fue el método de ensamblaje modular de ADN GoldenBraid (GB) basado en enzimas de restricción tipo IIS. Para optimizar el proceso de ensamblaje y catalogar la colección de fitobricks generados se desarrollaron una base de datos y un conjunto de herramientas software, tal y como se describe en el Capítulo 1. El paquete final de software se presentó en formato web como GB2.0, haciéndolo accesible al público a través de www.gbcloning.upv.es. El Capítulo 1 también proporciona una descripción detallada del funcionamiento de GB2.0 ejemplificando su uso con el ensamblaje de una construcción multigénica para la producción de antocianinas. Con el aumento en número y complejidad de las construcciones GB, el siguiente paso necesario fue el refinamiento de los estándar con la incorporación de la información experimental asociada a cada elemento genético (se describe en el Capítulo 2). Para este fin, el paquete de software de GB se reformuló en una nueva versión (GB3.0), un sistema de ensamblaje auto-contenido y completamente trazable en el que los datos experimentales que describen la funcionalidad de cada elemento genético se muestran en forma de una hoja de datos estándar. La utilidad de las especificaciones técnicas para anticipar el comportamiento de dispositivos biológicos compuestos se ejemplificó con la combinación de un interruptor químico y un prototipo de un módulo de sobreproducción de antocianinas equivalente al descrito en el Capítulo 1, resultando en un dispositivo de producción de antocianinas con respuesta a dexametasona. Además, en el Capítulo 3 se describe la adaptación a la tecnología GB de las herramientas de ingeniería genética CRISPR/Cas9, así como su caracterización funcional. La funcionalidad de estas herramientas para editado génico y activación y represión transcripcional se validó con el sistema de expresión transitoria en N.benthamiana. Finalmente, el Capítulo 4 presenta una implementación práctica del uso de la tecnología GB para hacer mejora vegetal de manera precisa. La transformación estable en tomate de una construcción intragénica que comprendía un marcador de selección intragénico y un regulador de la biosíntesis de flavonoides resultó en frutos con un mayor contenido de flavonoles. En conjunto, esta Tesis muestra la implementación de diseños genéticos cada vez más complejos y precisos en plantas utilizando elementos estándar y herramientas modulares siguiendo los principios de la Biología Sintética.[CA] La Biologia Sintètica és un camp emergent de caràcter interdisciplinar que es fonamenta amb l'aplicació a la enginyeria genètica dels principis de modularitat, abstracció i estandarització. Una nova vessant de la Biologia Sintètica aplicada a les plantes, la Biologia Sintètica Vegetal (BSV), ofereix noves possibilitats de millora de cultius que podrien portar a una millora de la resistència, a una major productivitat, o a un augment de la qualitat nutricional. Tanmateix, per poder arribar a este fi les eines moleculars disponibles en estos moments per a la BSV han d'adaptar-se per convertir-se en modulars, estàndards i més precises. Per això es plantejà com objectiu general d'aquesta Tesi adaptar, expandir i refinar les eines d'ensamblatge d'ADN de la BSV per permetre la incorporació d'especificacions funcionals en la descripció d'elements genètics estàndards (fitobricks) i facilitar la construcció d'estructures multigèniques cada vegada més complexes i precises, incloent eines d'edidat genètic. El punt de partida d'aquesta Tesi fou el mètode d'ensamblatge d'ADN modular GoldenBraid (GB) basat en enzims de restricció tipo IIS. Per optimitzar el proces d'ensamblatge i catalogar la col.lecció de fitobricks generats es desenvolupà una base de dades i un conjunt d'eines software, tal i com es descriu al Capítol 1. El paquet final de software es presentà en format web com GB2.0, fent-se accessible al públic mitjançant la pàgina web www.gbcloning.upv.es. El Capítol 1 també proporciona una descripció detallada del funcionament de GB2.0, exemplificant el seu ús amb l'ensamblatge d'una construcció multigènica per a la producció d'antocians. Amb l'augment en nombre i complexitat de les construccions GB, el següent pas fou el refinament dels estàndards amb la incorporació de la informació experimental associada a cada element genètic (es descriu en el Capítol 2). Per a aquest fi, el paquet de software de GB es reformulà amb una nova versió anomenada GB3.0. Aquesta versió consisteix en un sistema d'ensamblatge auto-contingut i complemtament traçable on les dades experimentals que descriuen la funcionalitat de cada element genètic es mostren en forma de fulla de dades estàndard. La utilitat de les especificacions tècniques per anticipar el comportament de dispositius biològics compostos s'exemplificà amb la combinació de un interruptor químic i un prototip d'un mòdul de sobreproducció d'antocians equivalent al descrit al Capítol 1. Aquesta combinació va tindre com a resultat un dispositiu de producció d'antocians que respón a dexametasona. A més a més, al Capítol 3 es descriu l'adaptació a la tecnologia GB de les eines d'enginyeria genètica CRISPR/Cas9, així com la seua caracterització funcional. La funcionalitat d'aquestes eines per a l'editat gènic i activació i repressió transcripcional es validà amb el sistema d'expressió transitòria en N. benthamiana. Finalment, al Capítol 4 es presenta una implementació pràctica de l'ús de la tecnologia GB per fer millora vegetal de mode precís. La transformació estable en tomaca d'una construcció intragènica que comprén un marcador de selecció intragènic i un regulador de la biosíntesi de flavonoïdes resultà en plantes de tomaca amb un major contingut de flavonols en llur fruits. En conjunt, esta Tesi mostra la implementació de dissenys genètics cada vegada més complexos i precisos en plantes utilitzant elements estàndards i eines modulars seguint els principis de la Biologia Sintètica.Vázquez Vilar, M. (2016). DESIGN OF GENETIC ELEMENTS AND SOFTWARE TOOLS FOR PLANT SYNTHETIC BIOLOGY [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68483TESISPremios Extraordinarios de tesis doctoralesCompendi