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At least 37 records · Page 2

Transformer Health Monitoring Using Dissolved Gas Analysis

As integral components of any power plant, transformers supply the generated electricity to the grid. However, a transformer’s cellulose-based paper insulation and the mineral oil in which it is immersed break down over time under standard operating conditions—or more rapidly due to potential faults within the system. As the transformer’s mineral oil breaks down, gases are released that can be measured and monitored. This technical brief exhibits a collection of diagnostic and prognostic techniques that utilities can adopt in lieu of labor-intensive periodic preventive maintenance routines. Furthermore, prognostic models have been incorporated using the latest version of the Institute of Electrical and Electronics Engineers (IEEE) standard (IEEE, 2019) for dissolved gas analysis (DGA), thus expanding it to include estimation of the time to maintenance. Overall, four different methodologies are explained, each of which aids in determining a transformer’s state of health. These methodologies include the Chendong model, the IEEE thermal life consumption model (IEEE, 2012), a diagnostic model for DGA, and a prognostic model for DGA that uses an autoregressive integrated moving average (ARIMA) model. An additional improvement for estimating missing system parameters by using monitoring data (i.e., a tool for parameter estimation utilizing Powell’s method) is presented, enabling the IEEE thermal life consumption model to benefit not only the collaborating power plant, but also the power industry at large.

24 POWER TRANSMISSION AND DISTRIBUTION↗

Multigene engineering in plants: Technologies, applications, and future prospects

The emerging bioeconomy presents a promising solution to both economic and environmental challenges. Within the bioeconomy, plants serve as a renewable, sustainable, and cost-effective source of foods, fuels, chemicals, and materials. However, traditional breeding and single-gene engineering approaches fall short in addressing complex traits (e.g., drought tolerance, disease resistance, yield, nutrient use efficiency) which are controlled by multiple genes. The complexity of plant biology often necessitates the use of multigene engineering (MGE), which involves simultaneous ectopic expression, up/down-regulation, or editing of multiple genes, to enhance plant traits relevant to the bioeconomy. These genes may be associated with distinct traits or function as components of specific metabolic and regulatory pathways. This review summarizes current technologies for MGE within the synthetic biology-driven Design-Build-Test-Learn (DBTL) framework, detailing its four key stages: Design – gene construct development; Build – DNA assembly and plant transformation; Test – the molecular, biochemical, and physiological characterization of engineered plants; and Learn – computational modeling to refine, multiplex and iterate the process. Despite good progress in the applications of MGE in biofortification, metabolic engineering, and stress resilience, challenges remain in construct stability, coordinated gene expression, and regulatory predictability. We identified optimization paths and future directions to accelerate MGE deployment in sustainable agriculture, with possible societal benefits including reduced production costs, increased yield, and improved food and nutritional security.

AI-aided plant engineering↗

Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid

Agrobacterium is not only a costly plant pathogen but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and complex transcriptional regulation make identification of the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here, we use a bottom-up synthetic approach to systematically engineer the tumor-inducing plasmid (pTi), wherein the majority of virulence machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the contributions of critical vir-genes at different expression levels. We construct a synthetic pTi capable of transient plant and stable fungal transformation and characterize bottlenecks and solutions for complex polygenic synthetic pTi designs. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, laying the foundation for future engineering to establish full synthetic control over the critical process of AMT.

Agrobacterium-mediated transformation↗

Through the lens of bioenergy crops: advances, bottlenecks, and promises of plant engineering

Advances in engineering of bioenergy crops were driven over the past years by adapting technological breakthroughs and accelerating conventional applications but also exposed intriguing challenges. New tools revealed rich interconnectivity in the exponentially growing and dynamic 'big' omics data' of metabolomes, transcriptomes, and genomes at previously inaccessible magnitude (global, cross-species, meta-) and resolution (single cell). Insights enabled fresh hypotheses and stimulated disciplines such as functional genomics with discovery of broad regulatory networks and their determinants, that is, DNA parts, including promoters, regulatory elements, and transcription factors. Their rational design, assembly into increasingly complex blueprints, and installation into diverse chassis is an existing frontier that may benefit from emerging technologies to address bottlenecks. Interweaving nature-inspired to fully synthetic parts has already allowed building of fine-tuned regulatory circuits, or new-to-nature metabolic routes insulated from the biological context of the chassis species. Similarly, developments and the evolving need for unifying principles in plant transformation and species-agnostic technologies highlight future opportunities for engineering the next generation of bioenergy plants.

60 APPLIED LIFE SCIENCES↗

Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high-yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence-derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock.

RUBY↗

Methods and compositions for enhanced biomass production and increased abiotic stress tolerance

This invention relates to methods for increasing carbon fixation, increasing biomass production and/or increasing abiotic stress tolerance in a plant comprising: introducing into a plant, plant part, and/or plant cell a heterologous polynucleotide encoding a ferredoxin polypeptide and/or a heterologous polynucleotide encoding a biotin ligase polypeptide to produce a stably transformed plant, plant part, and/or plant cell, wherein said heterologous polynucleotides are from a bacterial and/or an archaeal species. Further provided are plants, plant parts and plant cells produced by the methods of the invention.

Grunden, Amy Michele↗

Transformation and gene editing in the bioenergy grass Miscanthus

Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

09 BIOMASS FUELS↗

Plant Design for a Developing Bioeconomy Workshop Report: Frontier Science for the Bioeconomy Workshop Series

Recent advances in fundamental plant biology research, synthetic biology, and artificial intelligence (AI) are unlocking powerful new capabilities in plant biodesign, offering unprecedented potential to reimagine plants as programmable platforms for resource-efficient production of bioenergy, biomaterials, chemicals, and more. The U.S. Department of Energy (DOE) convened the Plant Design for a Developing Bioeconomy virtual workshop on March 12 through 14, 2025, to bring together leaders across plant science, engineering, and computation to assess the current landscape and define a bold vision for future research. Discussions during the workshop built upon findings included in DOE’s Biological and Environmental Research (BER) workshop report Overcoming Barriers in Plant Transformation: A Focus on Bioenergy Crops (U.S. DOE 2024; genomicscience. energy.gov/plant-transformation). Participants identified critical knowledge gaps, technical barriers, and emerging opportunities in the design and engineering of plant systems to support a robust, resilient domestic bioeconomy aligned with DOE’s mission.

09 BIOMASS FUELS↗

Plant science decadal vision 2020–2030: Reimagining the potential of plants for a healthy and sustainable future

Abstract Plants, and the biological systems around them, are key to the future health of the planet and its inhabitants. The Plant Science Decadal Vision 2020–2030 frames our ability to perform vital and far‐reaching research in plant systems sciences, essential to how we value participants and apply emerging technologies. We outline a comprehensive vision for addressing some of our most pressing global problems through discovery, practical applications, and education. The Decadal Vision was developed by the participants at the Plant Summit 2019, a community event organized by the Plant Science Research Network. The Decadal Vision describes a holistic vision for the next decade of plant science that blends recommendations for research, people, and technology. Going beyond discoveries and applications, we, the plant science community, must implement bold, innovative changes to research cultures and training paradigms in this era of automation, virtualization, and the looming shadow of climate change. Our vision and hopes for the next decade are encapsulated in the phrase reimagining the potential of plants for a healthy and sustainable future. The Decadal Vision recognizes the vital intersection of human and scientific elements and demands an integrated implementation of strategies for research (Goals 1–4), people (Goals 5 and 6), and technology (Goals 7 and 8). This report is intended to help inspire and guide the research community, scientific societies, federal funding agencies, private philanthropies, corporations, educators, entrepreneurs, and early career researchers over the next 10 years. The research encompass experimental and computational approaches to understanding and predicting ecosystem behavior; novel production systems for food, feed, and fiber with greater crop diversity, efficiency, productivity, and resilience that improve ecosystem health; approaches to realize the potential for advances in nutrition, discovery and engineering of plant‐based medicines, and "green infrastructure." Launching the Transparent Plant will use experimental and computational approaches to break down the phytobiome into a "parts store" that supports tinkering and supports query, prediction, and rapid‐response problem solving. Equity, diversity, and inclusion are indispensable cornerstones of realizing our vision. We make recommendations around funding and systems that support customized professional development. Plant systems are frequently taken for granted therefore we make recommendations to improve plant awareness and community science programs to increase understanding of scientific research. We prioritize emerging technologies, focusing on non‐invasive imaging, sensors, and plug‐and‐play portable lab technologies, coupled with enabling computational advances. Plant systems science will benefit from data management and future advances in automation, machine learning, natural language processing, and artificial intelligence‐assisted data integration, pattern identification, and decision making. Implementation of this vision will transform plant systems science and ripple outwards through society and across the globe. Beyond deepening our biological understanding, we envision entirely new applications. We further anticipate a wave of diversification of plant systems practitioners while stimulating community engagement, underpinning increasing entrepreneurship. This surge of engagement and knowledge will help satisfy and stoke people's natural curiosity about the future, and their desire to prepare for it, as they seek fuller information about food, health, climate and ecological systems.

59 BASIC BIOLOGICAL SCIENCES↗

Data for Transformation and Gene Editing in the Bioenergy Grass Miscanthus

Miscanthus, a C4 member of the family Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies Miscanthus x giganteus and its parental species M. sacchariflorus and M. sinensis . Use of biotechnology-based procedures to genetically improve miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites.

Biomass Analytics↗

SIVB's 2024 In Vitro Biology Meeting Proceedings

SIVB's 2024 World Congress on In Vitro Biology took place in Saint Louis, Missouri, from June 8th to 12th, 2024. The conference featured renowned speakers from academic and non-academic institutions who will present recent advancements in critical areas like plant transformation, genome editing, synthetic biology, advanced breeding technologies, cellular agriculture, future food sources, chromosome engineering, epigenetics, artificial intelligence, and machine learning. The Society for In Vitro Biology (SIVB) has always considered the education and professional development of young researchers as an integral component of its mission. The 2024 World Congress program, along with SIVB’s student initiatives, was customized to foster scientific growth and professional development among students and young scientists empowering them in their professional journeys. The recording of the DOE supported "Single Cell RNA Sequencing" workshop was made publicly available at https://youtu.be/A0UnuYwefwg for easy retrieval and reference of all information shared during the live event, thereby increasing accessibility and knowledge transfer. Their are 14 articles in the proceedings and the full list of files is located at https://link.springer.com/journal/11626/volumes-and-issues/60-1/supplement.

10 SYNTHETIC FUELS↗

In-vivo Raman microspectroscopy reveals differential nitrate concentration in different developmental zones in Arabidopsis roots

Abstract Background Nitrate (NO 3 − ) is one of the two major forms of inorganic nitrogen absorbed by plant roots, and the tissue nitrate concentration in roots is considered important for optimizing developmental programs. Technologies to quantify the expression levels of nitrate transporters and assimilating enzymes at the cellular level have improved drastically in the past decade. However, a technological gap remains for detecting nitrate at a high spatial resolution. Using extraction-based methods, it is challenging to reliably estimate nitrate concentration from a small volume of cells (i.e., with high spatial resolution), since targeting a small or specific group of cells is physically difficult. Alternatively, nitrate detection with microelectrodes offers subcellular resolution with high cell specificity, but this method has some limitations on cell accessibility and detection speed. Finally, optical nitrate biosensors have very good ( in-vivo ) sensitivity (below 1 mM) and cellular-level spatial resolution, but require plant transformation, limiting their applicability. In this work, we apply Raman microspectroscopy for high-dynamic range in-vivo mapping of nitrate in different developmental zones of Arabidopsis thaliana roots in-situ . Results As a proof of concept, we have used Raman microspectroscopy for in-vivo mapping of nitrate content in roots of Arabidopsis seedlings grown on agar media with different nitrate concentrations. Our results revealed that the root nitrate concentration increases gradually from the meristematic zone (~ 250 µm from the root cap) to the maturation zone (~ 3 mm from the root cap) in roots grown under typical growth conditions used for Arabidopsis, a trend that has not been previously reported. This trend was observed for plants grown in agar media with different nitrate concentrations (0.5–10 mM). These results were validated through destructive measurement of nitrate concentration. Conclusions We present a methodology based on Raman microspectroscopy for in-vivo label-free mapping of nitrate within small root tissue volumes in Arabidopsis. Measurements are done in-situ without additional sample preparation. Our measurements revealed nitrate concentration changes from lower to higher concentration from tip to mature root tissue. Accumulation of nitrate in the maturation zone tissue shows a saturation behavior. The presented Raman-based approach allows for in-situ non-destructive measurements of Raman-active compounds.

Fernández González, Alma↗

VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

59 BASIC BIOLOGICAL SCIENCES↗

Transcriptome architecture of the three main lineages of agrobacteria

ABSTRACT Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

59 BASIC BIOLOGICAL SCIENCES↗

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Weiss, Trevor↗

Improvements to PVWatts for Fixed and One-Axis Tracking Systems

This work presents improvements to the widely used NREL PVWatts photovoltaic system energy model to improve modeling accuracy for typical fixed and one axis system designs. The aim is to calculate losses in the PV system assuming typical modern system design practices, while maintaining simplicity by keeping the required set of input parameters small. These improvements allow users to more credibly and quickly evaluate competing system designs in early stage feasibility. Common submodels for module cover, spectral, snow, tracker, transformer, plant controller, and self-shading losses, in addition to a bifacial gain option, are incorporated into the PVWatts model, and are shown to improve PVWatts' system performance prediction capabilities without major impact to ease of use. We anticipate including these improvements in a future release of NREL's open source PVWatts code, and some of the features may become available in the System Advisor Model (SAM) desktop software as well as the popular PVWatts web application.

14 SOLAR ENERGY↗

Elucidating Mechanisms of Rust Pathogenesis for Engineering Resistance in Poplar

The overall objective of this research was to elucidate the molecular basis for the virulence of Melampsora larici-populina towards Populus spp. in order to address the formidable challenge of engineering durable resistance in poplar against leaf rust. Our original research plan was built on the technically innovative approach of using dual host systems and known host defense proteins and pathogen avirulent effectors along with genome-wide approaches to identify key pathogenesis effectors of Melampsora larici-populina that target poplar defense and nutrient acquisition. Our efforts focused on developing high-throughput experimental approaches and systems to enable us to address our overarching hypothesis that rust effectors with homology to other fungal effectors will either bypass the poplar immune system and/or suppress effector-triggered immunity through conserved mechanisms, and that an analysis of these interaction networks would provide for new approaches to develop rust-resistant poplar. Three main objectives were originally proposed: (1) mine key rust effectors that suppress host immunity; (2) elucidate interaction networks in poplar targeted by key rust effectors; (3) generate transgenic poplar resources for identification of genes involved in pathogen infection or host defense triggered by rust. Generally, we worked towards all three objectives by developing and applying complementary molecular, genomic, biochemical and plant transformation approaches.

54 ENVIRONMENTAL SCIENCES↗

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

Altpeter, Fredy (ORCID:0000000208944976)↗