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Abstract High‐precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR‐associated 9) system is a programmable, RNA‐guided nuclease that introduces targeted, site‐specific double‐stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame‐shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step‐by‐step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in‐house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2:Agrobacteriumtransformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events 

SUMMARY Plant genetic transformation is essential for understanding gene functions and developing improved crop varieties. Traditional methods, often genotype‐dependent, are limited by plants' recalcitrance to gene delivery and low regeneration capacity. To overcome these limitations, new approaches have emerged that greatly improve efficiency and genotype flexibility. This review summarizes key strategies recently developed for plant transformation, focusing on groundbreaking technologies enhancing explant‐ and genotype flexibility. It covers the use of morphogenic regulators (MRs), stem cell‐based methods, andin plantatransformation methods. MRs, such as maizeBabyboom(BBM) withWuschel2(WUS2), andGROWTH‐REGULATING FACTORs(GRFs) with their cofactorsGRF‐interacting factors(GIFs), offer great potential for transforming many monocot species, including major cereal crops. OptimizingBBM/WUS2expression cassettes has further enabled successful transformation and gene editing using seedling leaves as starting material. This technology lowers the barriers for academic laboratories to adopt monocot transformation systems. For dicot plants, tissue culture‐free orin plantatransformation methods, with or without the use of MRs, are emerging as more genotype‐flexible alternatives to traditional tissue culture‐based transformation systems. Additionally, the discovery of the local wound signal peptide Regeneration Factor 1 (REF1) has been shown to enhance transformation efficiency by activating wound‐induced regeneration pathways in both monocot and dicot plants. Future research may combine these advances to develop truly genotype‐independent transformation methods. 

Abstract The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro‐propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype‐independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype‐flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype‐flexible morphogenic gene‐based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well‐established, work‐horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research. 

Summary The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plantsin vitrohas been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation‐based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non‐responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the geneWox2awas found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences ofWox2awere found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in theWox2aoverexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response toWox2a, our data support the utility ofWox2ain enabling transformation of recalcitrant genotypes. 

Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability madeAgrobacteriuman essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such asAgrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system forAgrobacteriumgenome engineering. In this work, we demonstrate thatINsertion ofTransposableElements byGuideRNA–AssistedTargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxPrecombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies forAgrobacteriumspecies and their gene functional analyses. 

The ability to precisely engineerAgrobacteriumstrains is crucial for advancing their utility in plant biotechnology. We recently implemented the CRISPR RNA-guided transposase system, INTEGRATE, as an efficient tool for genetic modification inAgrobacterium. Despite its promise, the practical application of INTEGRATE inAgrobacteriumstrain engineering remains underexplored. Here, we present a standardized and optimized workflow that enables researchers to harness INTEGRATE for targeted genome modifications. By addressing common challenges, such as crRNA design, transformation efficiency, and vector eviction, this protocol expands the genetic toolkit available forAgrobacterium, facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticatedAgrobacterium rhizogenesK599 strain by deleting the 15-kb T-DNA region from its root-inducing plasmid pRi2659 and inactivating a thymidylate synthase gene to render the strain auxotrophic for thymidine. The protocol provides detailed guidance for each step, including target site selection, crRNA spacer cloning,Agrobacteriumtransformation, screening for targeted insertion and Cre/loxP-mediated deletion, and vector removal. This resource will empower new users to perform efficient and reproducible genome engineering inAgrobacteriumusing the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology. 

Chromosome architecture plays a crucial role in bacterial adaptation, yet its direct impact remains unclear. Different bacterial species and even strains within the same species exhibit diverse chromosomal configurations, including a single circular or linear chromosome, two circular chromosomes, or a circular-linear combination. To investigate how these architectures shape bacterial behavior, we generated near-isogenic strains representing each configuration inAgrobacterium tumefaciensC58, an important soil bacterium widely used for plant genetic transformation. Strains with a single-chromosome architecture, whether linear or circular, exhibited faster growth, enhanced stress tolerance, and greater interstrain competitiveness. In contrast, bipartite chromosome strains showed higher virulence gene expression and enhanced transient plant transformation efficiency, suggesting a pathogenic adaptation. Whole-transcriptome analysis revealed architecture-dependent gene expression patterns, underscoring the profound impact of chromosome organization onAgrobacteriumfitness and virulence. These findings highlight how chromosome structure influences bacterial adaptation and shapes evolutionary trajectories. 

Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additionalvirgene helper plasmid into disarmedAgrobacteriumstrains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophicAgrobacteriumstrains to boostAgrobacterium-mediated plant transformation efficiency. AuxotrophicAgrobacteriumstrains are useful in reducingAgrobacteriumovergrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from publicAgrobacteriumstrains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transientGUSexpression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries thevirAgene from pTiBo542 in addition to othervirgene operons (virG,virB,virC,virD,virE, andvirJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improvedAgrobacteriumsystem, including auxotrophic disarmedAgrobacteriumstrains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.