Plant Biotechnology Journal

We developed a non-culture and DNA-free genome editing technique for melon using in plant Particle Bombardment (iPB-RNP). The method was employed to create CmACO1 mutants. One of the mutant lines exhibited an extended shelf life due to ethylene deficiency during fruit ripening. As no cell culture step is involved, the iPB-RNP method is expected to overcome limitations associated with conventional genome editing, such as genotype dependency and somatic variations. Thus, this methodology holds significant potential for application in commercial melon breeding and across a wide range of Cucurbitaceae species.

Genome editing technologies possess significant potential to enhance plant breeding; however, the delivery of editing constructs poses challenges in numerous crop species due to their resistance to transformation or tissue culture. This study introduces an innovative approach for the delivery of ribonucleoprotein (RNP) complexes and plasmid vectors into intact, regenerable faba bean plant tissues. This is accomplished by applying an electric current that makes plant cell walls and membranes permeable, thereby allowing the entry of macromolecular constructs into the cell and nucleus. This study assessed the efficacy of electric pulse-mediated transfection in faba bean by generating stable GFP-expressing faba bean plants. Furthermore, we incorporated it into faba bean leaf tissue and demonstrated its application in both embryos and leaf tissues. We demonstrate DNA-free genome editing by targeting the endogenous phytoene desaturase gene (PDS), achieving a mutation success rate of 50%. This method is efficient and economical, necessitating limited technical training. It is applicable to both leaf and embryo tissues, thereby enhancing its utility for crop improvement. This technique shows potential for the development of new crop varieties that can more effectively address global climate challenges.

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system, that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we primarily generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knockingout the first betalain biosynthetic gene in RUBY-positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods.

RNA interference (RNAi) is a highly conserved gene silencing mechanism regulating gene expression at transcriptional and post-transcriptional levels in plants. Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) have emerged as powerful tools for highly specific and efficient gene silencing. However, their application in crops has been constrained by the need for transgene integration and the relatively long length of TAS-derived precursors. Here, we developed a novel syn-tasiRNA platform for Solanum lycopersicum (tomato) based on minimal precursors targeted by endogenous SlmiR482b microRNA. These minimal precursors, comprising only a 22-nt miRNA target site, an 11-nt spacer and the syn-tasiRNA sequence(s), effectively produced functional syn-tasiRNAs in both transgenic and transient virus-induced gene silencing (syn-tasiR-VIGS) systems. To facilitate their broader application, we engineered a series of vectors for high-throughput cloning and efficient syn-tasiRNA expression from SlmiR482b-based minimal precursors in tomato. Our results show that minimal precursors induce robust gene silencing of endogenous tomato genes and confer antiviral resistance to the economically important tomato spotted wilt virus. Furthermore, we show that syn-tasiR-VIGS can be applied in a transgene-free manner through crude extract delivery, leading to efficient silencing of endogenous genes. This study establishes minimal syn-tasiRNA precursors as a versatile and efficient tool for precision RNAi in tomato, with applications in functional genomics and crop improvement.

Efficient transformation remains a major constraint to functional genomics and genome editing in Vaccinium, where stable transformation systems are often genotype-dependent and inefficient. Here, we establish a rapid and high-efficiency Rhizobium rhizogenes-mediated hairy root transformation platform optimized for the genus. Using the RUBY visual reporter, transformation efficiency reached 46.7% in leaf explants infected with strain Ar. A4 and cultured on half-strength Woody Plant Medium, with transgenic roots visible within 16 days post-co-cultivation. Comparative evaluation of six R. rhizogenes strains identified Ar. A4 and ATCC15834 as consistently superior across diverse Vaccinium germplasm representing different taxonomic sections, achieving up to 80% efficiency in selected genotypes. While conventional regeneration from transgenic roots was not successful, overexpression of developmental regulators enabled shoot formation with 7% efficiency, demonstrating a path toward stable plant recovery. This platform delivers a rapid, genotype-flexible system for gene validation, metabolic pathway analysis, and genome editing in Vaccinium, substantially expanding the molecular toolkit available for perennial fruit crop research and translational breeding.

Plant-derived pigments offer sustainable alternatives to synthetic colorants, yet their practical deployment in textiles is limited by restricted chemical diversity and low abundance. Liverworts represent a source of diverse chemical compounds, and the model liverworts Marchantia polymorpha is an emerging as chassis for bioengineering and synthetic biology. Here, we report the biotechnological application of auronidins, a rare class of flavonoid pigments, as textile dyes. Using the Marchantia, we engineered enhanced auronidin production through controlled expression of the R2R3-MYB transcription factor MpMYB14. We systematically benchmarked constitutive and inducible gene expression systems, including heat-shock, glucocorticoid receptor, and {beta}-estradiol (XVE) circuits, identifying inducible strategies that decouple biomass accumulation from secondary metabolite production while achieving high pigment yields. Extracted auronidins were used to dye cotton yarn directly, demonstrating the feasibility of auronidins for textile dyeing. Our results establish Marchantia as a versatile plant chassis for programmable secondary metabolite production and introduce auronidins as a promising natural pigment platform for sustainable textile biotechnology.

There is a growing impetus to expand the repository of chassis available to synthetic biologists. The chloroplast genome presents a unique chassis for engineering photosynthetic eukaryotes by virtue of its compact size, lack of epigenetic regulation, and containment within the secluded lipid bilayers of the organelle. The development of the chloroplast as a synthetic biology chassis, however, has been limited by a lack of efficient techniques for whole genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117 kb Phaeodactylum tricornutum chloroplast genome that have 90 to 100% efficiency when screening as few as ten Saccharomyces cerevisiae colonies following yeast assembly. The first method directly uses PCR-amplified fragments of the genome for yeast assembly, whereas the second method relies upon the pre-cloning of eight overlapping genomic regions into individual plasmids that they can later be released from. The cloned genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a novel delivery mechanism for bringing DNA directly to the algal chloroplast. As well, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly.

The lack of resistance resources is a major bottleneck for wheat Fusarium head blight (FHB) resistance breeding. Three wheat-Th. elongatum FHB resistant translocation lines have been developed and used for wheat breeding without yield penalty. Transcriptomic analysis identified a derivative glutathione S-transferase transcript T26102, which was homologous to Fhb7 and induced dramatically by Fusarium graminearum. Unlike other studies, Fhb7 homologs were detected not only in Thinopyrum but also in Elymus, Leymus, Pseudoroegeria and Roegeria. We also found that several wheat-Th. ponticum derivatives carrying Fhb7 and its homologs were highly susceptible to FHB. Moreover, the transgenic plants expressing Fhb7 and its homolog on different backgrounds did not improve the FHB resistance. One Sentence SummaryThe GST-encoding Fhb7 candidate cannot improve Fusarium head blight resistance in wheat breeding.

Stripe rust, a globally widespread disease, stands as one of the most significant threats to wheat cultivation. The 1BL/1RS translocation, renowned for its robust resistance to both rust and powdery mildew, has historically played an important role in wheat breeding and production. The gene for resistance to stripe rust on the 1RS is known as Yr9 and plays an important role in the production of wheat, but over the course of long-term breeding had lost its resistance due to the evolution of stripe rust towards greater and greater virulence. In this paper, we cloned the stripe rust resistance gene, Yr9, from triticale by genetic mapping approach. The Yr9 encodes a typical nucleotide-binding leucine-rich repeat (NLR) protein. Both transgenic and overexpression of Yr9 in highly stripe rust susceptible wheat varieties conferred complete resistance to the stripe rust races CYR17 and partial resistance to the stripe rust races CYR32, CYR33, and CYR34. In addition, the Yr9 allele in the 1BL/1RS translocation line also showed the same level of resistance to stripe rust. Both two alleles loses resistance when deployed in the field or inoculated with mixed physiological races collected from the field. Our findings provide valuable insights for breeders to strategically incorporate disease resistance genes and provides a foundation for further understanding how pathogenic bacteria might evolve to evade recognition via NLR type proteins. SignificanceThe 1BL/1RS translocation between wheat and rye is the most successful case of exogenous gene application in plant genetic improvement and has been used in wheat breeding for over 50 years. Here we report the cloning of a stripe rust resistance gene Yr9 located on rye chromosome 1RS using a triticale population. The Yr9 encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein that show complete resistance to the stripe rust races CYR17 and partial resistance to the stripe rust races CYR32, CYR33, and CYR34, albeit demonstrating susceptibility under field conditions. Our findings position Yr9 as an ideal candidate gene to study the mechanism of inactivation of disease resistance genes as a result of pathogen evolution.

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

Strigolactones (SLs) are plant hormones regulating shoot branching and symbiotic interactions, but their trace-level abundance limits research and applications. Here, we optimized a Nicotiana benthamiana transient expression system for SL production by tuning agroinfiltration parameters and co-expressing rate-limiting carotenoid biosynthetic genes. Overexpression of Zea mays PSY1 or an Arabidopsis PSY-GGPS11 fusion increased carlactone production over 2-fold and enhanced downstream SL accumulation. Using this platform, we discovered that sorghum cytochrome P450 SbCYP728B35 catalyzes conversion of 5-deoxystrigol to sorgolactone, revealing a previously unknown function. These results establish metabolic engineering of precursor supply as an effective strategy for boosting SL production and demonstrate N. benthamiana as a robust system for pathway elucidation and biotechnological synthesis of bioactive strigolactones.

Terpenes constitute the largest and most structurally diverse class of plant secondary metabolites, with critical roles in plant-environment interactions and broad industrial applications. Although nuclear genome engineering of terpene pathways has been extensively explored, chloroplast genome engineering remains largely undeveloped, with all reported studies restricted to the model plant Nicotiana. Here we report successful chloroplast genome engineering for diterpene production in the crop plant potato (Solanum tuberosum). First we identified the trnT/trnL plastomic locus as optimal for minimizing integration-associated growth penalties. Insertion of a bifunctional diterpene synthase gene into this plastomic site yielded transplastomic plants with successful diterpene production, but with reduced growth. The co-expression of a geranylgeranyl diphosphate synthase gene to enhance precursor supply restored normal growth while elevating diterpene accumulation. Transplastomic plants were otherwise agronomically comparable to wild-type. This work expands chloroplast engineering as a viable strategy for terpene pathway engineering in crop improvement and high-value terpene production.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explore the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice genes. We find that SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE method. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.

The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with this tissue culture-free genome editing platform, opens up possibilities for editing across a wide range of plant species.

Tea is the oldest and most popular nonalcoholic beverage consumed in the world. It provides abundant secondary metabolites that account for its diverse flavors and health benefits. Here we present the first high-quality chromosome-length reference genome of C. sinensis var. sinensis using long read single-molecule real time (SMRT) sequencing and Hi-C technologies to anchor the [~]2.85-Gb genome assembly into 15 pseudo-chromosomes with a scaffold N50 length of [~]195.68 Mb. We annotated at least 2.17 Gb ([~]74.13%) of repetitive sequences and high-confidence prediction of 40,812 protein-coding genes in the [~]2.92-Gb genome assembly. This accurately assembled genome allows us to comprehensively annotate functionally important gene families such as those involved in the biosynthesis of catechins, theanine and caffeine. The contiguous genome assembly provides the first view of the repetitive landscape allowing us to accurately characterize retrotransposon diversity. The large tea tree genome is dominated by a handful of Ty3-gypsy long terminal repeat (LTR) retrotransposon families that recently expanded to high copy numbers. We uncover the latest bursts of numerous non-autonomous LTR retrotransposons that may interfere with the propagation of autonomous retroelements. This reference genome sequence will largely facilitate the improvement of agronomically important traits relevant to the tea quality and production.

Precise, site-specific integration of large DNA sequences into plant genomes is a cornerstone of crop biotechnology and synthetic biology, yet remains constrained by random insertion, inefficient homologous recombination, and gene targeting. Here, we present PrimeStack, a DSB-independent platform that integrates prime editing with the unidirectional large serine integrase Bxb1, leveraging evolved variants for enhanced activity, to achieve the programmable insertion of multigene, multikilobase cargos at predefined genomic safe harbors in rice. Optimized prime editors first install attP landing sites with high fidelity and heritability followed by Bxb1-mediated recombination that generates irreversible integration of genetic information. PrimeStack achieves integration frequencies of approximately 43-46% (as detected by junction-specific PCR in rice calli), with phenotypic neutrality in regenerated plants, comparing favorably with bidirectional Cre-lox systems. We validate its utility by achieving targeted insertion of a carotenoid cassette at an experimentally confirmed genomic safe harbor. PrimeStack delivers a modular, site-specific gene-stacking platform that enables predictable, multigene trait pyramiding and pathway construction at genomic safe harbors, thereby accelerating the development of improved and resilient crop varieties, as well as scalable plant-based biomanufacturing and a powerful chassis for synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/718181v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@59c915org.highwire.dtl.DTLVardef@a0ba4corg.highwire.dtl.DTLVardef@26f8d4org.highwire.dtl.DTLVardef@9a3efe_HPS_FORMAT_FIGEXP M_FIG C_FIG

The semi dwarf stature of modern wheat varieties is conferred by RHT1 alleles derived from a single Japanese cultivar. These alleles are absent in landrace collections such as the Watkins collection. This constrains the direct use of rich genetic diversity preserved in Watkins landraces. These ancestral accessions, adapted to diverse local environments, harbour valuable traits absent from elite cultivars. Here, we demonstrate a precision breeding approach that integrates CRISPR/Cas9, cytosine base editing, and prime editing to introduce semi-dwarfing alleles into selected Watkins landraces. Our strategy overcomes problems caused by the tall stature of most Watkins accessions, providing rapid and precise modification of the Rht1 locus to confer semi-dwarf phenotypes. High editing efficiencies achieved across multiple Watkins landrace wheat lines confirm the robustness of our approach. By unlocking previously untapped genetic variation and enabling targeted trait integration, this study lays the foundation for modern landrace-based breeding programs, supporting sustainable wheat improvement and global food security.

Monoterpene indole alkaloids (MIAs) are a major class of plant natural products with important pharmaceutical activities, yet the biosynthetic pathway to their universal precursor, strictosidine, has been fully elucidated in only Catharanthus roseus. In kratom (Mitragyna speciosa), only the first and last steps of strictosidine biosynthesis were previously known. Here, we applied multiplex pathway engineering in yeast to accelerate the discovery, reconstruction, and optimization of the kratom strictosidine pathway. Iterative multiplex integration and screening identified 13 functional kratom genes and enabled rapid validation of functional pathway modules, thereby completing the kratom strictosidine pathway from geranyl pyrophosphate and tryptophan. We also identified a vacuolar secologanin transporter, MsNPF2.6, which increased strictosidine production by 62% in yeast. Pathway optimization through the incorporation of nepetalactol-producing enzymes from other plants further supported strictosidine production in yeast from fed geraniol and tryptophan. These results establish the strictosidine pathway in kratom and highlight multiplex engineering as a powerful platform for rapid plant pathway discovery and optimization.