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11 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Woody Plant Transformation: Current Status, Challenges, and Future Perspectives

Woody plants, comprising forest and fruit tree species, provide essential ecological and economic benefits to society. Their genetic improvement is challenging due to long generation intervals and high heterozygosity. Genetic transformation, which combines targeted DNA delivery with plant regeneration from transformed cells, offers a powerful alternative to accelerating their domestication and improvement. Agrobacterium tumefaciens, Rhizobium rhizogenes, and particle bombardment have been widely used for DNA delivery into a wide variety of explants, including leaves, stems, hypocotyls, roots, and embryos, with regeneration occurring via direct organogenesis, callus-mediated organogenesis, somatic embryogenesis, or hairy root formation. Despite successes, conventional approaches are hampered by low efficiency, genotype dependency, and a reliance on challenging tissue culture. This review provides a critical analysis of the current landscape in woody plant transformation, moving beyond a simple summary of techniques to evaluate the co-evolution of established platforms with disruptive technologies. Key advances among these include the use of developmental regulators to engineer regeneration, the rise in in planta systems to bypass tissue culture, and the imperative for DNA-free genome editing to meet regulatory and public expectations. By examining species-specific breakthroughs in key genera, including Populus, Malus, Citrus, and Pinus, this review highlights a paradigm shift from empirical optimization towards rational, predictable engineering of woody plants for a sustainable future.

Agrobacterium tumefaciens↗
DOE OSTI2024

Final Technical Report

The Department of Energy is interested in technologies that support the sustainable production of fuels, chemicals, and other bioproducts from plant biomass, to offset the nation’s reliance on fossil resources. The plant cell wall of energy crops provides the largest reservoir of raw materials for bioproducts. However, the widespread use of plant cell walls is hampered by their complexity and resistance to breakdown. To improve the productivity and cost-effectiveness of using energy crops to generate bioproducts, the fundamental problem of deconstructing plant cell walls must be addressed. This project developed and evaluated an innovative genetic modification technology to produce strategically designed enzymes that specifically accumulate in the plant cell wall. The resulting enzyme-engineered energy crops are expected to grow normally under natural conditions but break down more quickly and easily under high temperature during the production of biobased products. As such, this plant cell wall targeting enzyme engineering effort will reduce the cost of plant cell wall deconstruction and ultimately improve the economics of bioproducts. The overall objective of this project is to develop and evaluate the in-planta enzyme engineering technology to reduce lignocellulose deconstruction cost. The concept was first validated using tobacco plant, a model plant system that is typically used in lab testing for initial concept validation. Then the enzyme optimization was validated using switchgrass, the energy crop to be used to produce bioproducts. There are three specific objectives in this Phase I project: (1) validate the enzyme optimization concept using tobacco plant, a model plant system. (2) validate the enzyme optimization concept using switchgrass. (3) techno-economic analysis (TEA) for further scale-up application. By the end of this project, in-planta enzyme engineering was validated in both tobacco and switchgrass plants, with improved enzyme activity and saccharification efficiency. The in-planta enzyme engineering in Tabacco didn’t have a significant impact on plant growth and development. Transgenic tobacco plants with in-planta cellulose degrading enzymes showed higher biomass digestibility than wild type. Gene construction and transformation in switchgrass was much longer than expected, which delayed the research progress. Besides, in-planta engineering of lignin degrading enzyme is more challenging than cellulose degrading enzyme, in terms of expression detection. Expression of lignin degrading enzyme and cellulose degrading enzyme improved biomass yield and saccharification efficiency of switchgrass, respectively. It is promising to express both genes in switchgrass for optimized overall performance. According to the results of TEA, switchgrass biomass production cost is mainly attributed to by fertility and harvesting. Biomass production profit can increase up to 10-fold depending on biomass price. The PHA production profit is also sensitive to the biomass price. The proposed technology could potentially reduce the biomass deconstruction cost from 33% to 9% of PHA revenue, making the biomass-based PHA competitive to petroleum-based polymers even in case of relatively high biomass price of biomass. Therefore, cultivation of the genetically engineered self-deconstruction switchgrass for Polyhydroxyalkanoate (PHA) production could benefit switchgrass grower and PHA producer with attractive profits for both sectors. This new enzyme optimization approach will be beneficial for bioindustries that use energy crops as feedstocks. It will improve the economic viability of converting energy crops to renewable products that support a sustainable society and helps address the Nation’s long-term strategic needs for renewable products and reduction of reliance on fossil resources.

42 ENGINEERING↗
DOE OSTI2022

Identification and characterization of a set of monocot BAHD monolignol transferases

Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as important “monomers” for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

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↗
DOE OSTI2026

BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana

Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission. Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

barley↗
DOE OSTI2021

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↗
DOE OSTI2024

GWAS supported by computer vision identifies large numbers of candidate regulators of in planta regeneration in Populus trichocarpa

Plant regeneration is an important dimension of plant propagation and a key step in the production of transgenic plants. However, regeneration capacity varies widely among genotypes and species, the molecular basis of which is largely unknown. Association mapping methods such as genome-wide association studies (GWAS) have long demonstrated abilities to help uncover the genetic basis of trait variation in plants; however, the performance of these methods depends on the accuracy and scale of phenotyping. To enable a large-scale GWAS of in planta callus and shoot regeneration in the model tree Populus, we developed a phenomics workflow involving semantic segmentation to quantify regenerating plant tissues over time. We found that the resulting statistics were of highly non-normal distributions, and thus employed transformations or permutations to avoid violating assumptions of linear models used in GWAS. We report over 200 statistically supported quantitative trait loci (QTLs), with genes encompassing or near to top QTLs including regulators of cell adhesion, stress signaling, and hormone signaling pathways, as well as other diverse functions. Our results encourage models of hormonal signaling during plant regeneration to consider keystone roles of stress-related signaling (e.g. involving jasmonates and salicylic acid), in addition to the auxin and cytokinin pathways commonly considered. The putative regulatory genes and biological processes we identified provide new insights into the biological complexity of plant regeneration, and may serve as new reagents for improving regeneration and transformation of recalcitrant genotypes and species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Enabling Lignin Valorization Through Integrated Advances in Plant Biology and Biorefining

Despite lignin having long been viewed as an impediment to the processing of biomass for the production of paper, biofuels, and high-value chemicals, the valorization of lignin to fuels, chemicals, and materials is now clearly recognized as a critical element for the lignocellulosic bioeconomy. However, the intended application for lignin will likely require a preferred lignin composition and form. To that end, effective lignin valorization will require the integration of plant biology, providing optimal feedstocks, with chemical process engineering, providing efficient lignin transformations. Recent advances in our understanding of lignin biosynthesis have shown that lignin structure is extremely diverse and potentially tunable, while simultaneous developments in lignin refining have resulted in the development of several processes that are more agnostic to lignin composition. Here, we review the interface between in planta lignin design and lignin processing and discuss the advances necessary for lignin valorization to become a feature of advanced biorefining.

09 BIOMASS FUELS↗
DOE OSTI2024

MultiGreen: A multiplexing architecture for GreenGate cloning

Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation—which is also its greatest flaw. MultiGreen seeks to address GreenGate’s limitations while maintaining the syntax of the original GreenGate kit. The primary limitations MultiGreen addresses are 1) multiplexing in series, 2) multiplexing in parallel, and 3) repeated cycling of transcriptional unit assembly through binary intermediates. MultiGreen efficiently concatenates bespoke transcriptional units using an additional suite of level 1acceptor vectors which serve as an assembly point for individual transcriptional units prior to final, level 2, condensation of multiple transcriptional units. Assembly with MultiGreen level 1 vectors scales at a maximal rate of 2*⌈ log 6 n ⌉+3 days per assembly, where n represents the number of transcriptional units. Further, MultiGreen level 1 acceptor vectors are binary vectors and can be used directly for plant transformation to further maximize prototyping speed. MultiGreen is a 1:1 expansion of the original GreenGate architecture’s grammar and has been demonstrated to efficiently assemble plasmids with multiple transcriptional units. MultiGreen has been validated by using a truncated violacein operon from Chromobacterium violaceum in bacteria and by deconstructing the RUBY reporter for in planta functional validation. MultiGreen currently supports many of our in-house multi transcriptional unit assemblies and will be a valuable strategy for more complex cloning projects.

Science & Technology - Other Topics↗
DOE OSTI2026

Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k cat,air /K c,air ) under ambient conditions, driven by a significantly faster turnover rate (k cat,air ). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.

59 BASIC BIOLOGICAL SCIENCES↗