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PubMed2026

Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.

Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.

Plant Viruses↗
PubMed2026

Genetic transformation of forage crops: comparative barriers, evidence, and emerging strategies.

Forage crops include phylogenetically and biologically distinct legumes and grasses, and their genetic transformation is constrained by different combinations of host response, DNA-delivery efficiency, regeneration competence, genotype dependence, and genome stability. This review critically compares evidence from forage legumes and forage grasses rather than treating these groups as a single transformation category. We evaluate Agrobacterium-mediated transformation, protoplast-based delivery, particle bombardment, CRISPR/Cas-enabled applications, developmental regulators (DRs), viral vectors, and nanomaterial-mediated delivery according to four practical outcomes: reproducibility across genotypes, recovery of regenerated plants, heritable transmission, and genetic stability. Direct evidence in forage crops shows that protocol performance is strongly species-, genotype-, explant-, and endpoint-dependent; efficiencies based on transient reporters or resistant callus therefore cannot be directly equated with stable, fertile events. DR-assisted regeneration has direct proof of concept in recalcitrant forage grasses, whereas stable nanomaterial-mediated transformation and virus-induced heritable editing remain unvalidated in forage crops. We conclude that current progress is best interpreted as the engineering of interacting delivery and regeneration constraints, not as a universal transition to genotype-independent transformation. Priority should be given to standardized outcome reporting, multi-genotype and inter-laboratory validation, controlled DR expression, and rigorous molecular and phenotypic assessment of regenerated plants.

Crops, Agricultural↗
PubMed2025

Uncovering the genetic basis of competitiveness and the potential for cooperation in plant groups.

Crop productivity was transformed by incorporating dwarfing genes that made plants smaller and less competitive (more cooperative). Beyond such major shifts in plant size, however, it is not clear how much variation in competitiveness remains and how to find its genetic basis. We performed plant density experiments, using 484 lines of the Arabidopsis thaliana multi-parent advanced generation inter-cross population, to compare methods for mapping the genetic basis of plant competitiveness. We first found that a major dwarfing gene, the erecta allele, caused reduced competitiveness and higher group productivity. Then, measuring competitiveness more generally, we found: (i) extensive variation in generic measures of competitiveness that extended beyond the effects of the erecta allele; (ii) a novel genomic region underlying variation in competitiveness; and (iii) that some measures of competitiveness were more useful than others. Our results show how modern genomic resources, including multi-parent populations, could uncover hidden genes for more cooperative crop plants.

Arabidopsis↗
PubMed2026

Controlling GRF4-GIF1 expression for efficient, genotype-independent transformation across wheat cultivars.

Wheat is a staple crop critical for global food security, and its continuous genetic improvement is essential to meet the demands of a growing population. Efficient, genotype-independent transformation is a major bottleneck in wheat functional genomics and gene editing. The growth regulating factor (GRF)-GRF-interacting factor (GIF) fusion technology enhances regeneration efficiency and broadens the range of transformable cultivars, but constitutive expression can reduce fertility and spikelet number. Here, we present an optimised Agrobacterium-mediated wheat transformation protocol incorporating GRF4-GIF1, tested across multiple tetraploid and hexaploid cultivars. Transformation efficiency was improved through adjustments in selection pressure, zeatin concentration, and promoter choice, with GRF4-GIF1 consistently enabling successful transformation across genotypes. Tissue-specific promoters and heat-inducible excision strategies effectively minimised pleiotropic effects, such as reduced fertility, while maintaining high transformation rates. This refined system provides a robust and versatile platform for gene function studies and gene editing, advancing genotype-independent wheat transformation and supporting breeding efforts to improve crop productivity, resilience, and nutritional value.

Triticum↗
PubMed2026

Genome Editing in Solanaceae: Harnessing CRISPR-Cas Technology for Precision Crop Improvement.

Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience.

CRISPR/Cas↗
PubMed2025

An elegant co-transformation strategy for recalcitrant wheat using morphogenic regulators.

Common wheat (Triticum aestivum L.) is a vital global crop, but many elite cultivars remain recalcitrant to genetic transformation, hindering functional genomics and crop improvement. Here, we developed an efficient co-transformation strategy for recalcitrant wheat varieties (e.g., Aikang58 and Xinong979) using the morphogenic gene mTaGRF4-TaGIF1. This approach entails mixing Agrobacterium tumefaciens cultures carrying two separate vectors: a standard gene-of-interest (GOI) vector (containing a selectable marker) and a gene-of-co-transformation vector (GOC, expressing mTaGRF4-TaGIF1 without a selectable marker). Co-transformation enhanced regeneration efficiency to ~37.38% in AK58, a marked improvement over conventional methods, enabling consistent recovery of transgenic plants. Among regenerants, ~63.25% carried both GOI and GOC (GOI&GOC), while ~11.92% contained only the GOI. Only-GOI plants could also be obtained through progeny segregation from GOI&GOC lines. We successfully generated GUS- and RUBY-expressing transgenic lines, as well as CRISPR-Cas9-edited mutants targeting Q and Ph1 genes, confirming the method's efficacy for both gain-of-function and genome editing application. Furthermore, the strategy was successfully extended to another recalcitrant variety Xinong979, demonstrating its potential for broad applicability. Unlike existing methods dependent on complex excision systems or tissue-specific promoters, our co-transformation methodology significantly simplifies both vector design and procedural workflow while maintaining high efficiency. Collectively, these findings establish a technically advanced yet operationally simplified transformation platform that addresses the long-standing challenge of genetic transformation in recalcitrant wheat varieties, providing researchers with a powerful tool for functional genomics studies and accelerating precision breeding programs in elite wheat cultivars.

Triticum↗
PubMed2026

Rapid Agrobacterium-mediated transformation and high-efficiency regeneration of finger millet (Eleusine coracana) for crop improvement.

Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens-mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5 mg L⁻1 6-benzylaminopurine (BAP), 1.5 mg L⁻1 kinetin, 0.1 mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2 mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30-32% were achieved in PR-202 and VL-376, respectively, by optimising infection and co-cultivation conditions, including reduced MS salt strength and pre-incubation of Agrobacterium. Molecular analyses, including PCR and Southern blot hybridisation, confirmed stable T-DNA integration in independent lines, while segregation analysis of T₁ progenies demonstrated Mendelian inheritance of the transgene. In addition, CRISPR/Cas9 constructs targeting EcCKX2 were successfully introduced via Agrobacterium, demonstrating the suitability of this system for genome engineering applications. Overall, this optimised SAM-based protocol provides a rapid (45-50 days), efficient, and reproducible platform for stable genetic transformation in finger millet and establishes a strong foundation for transgenic research and future genome editing studies in this underutilized crop.

Eleusine↗
PubMed2026

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.

The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.

Gene Editing↗
PubMed2026

Plant-derived and microbial biostimulants in sustainable agriculture: mechanisms, applications, and challenges.

Plant biostimulants have emerged as transformative and sustainable tools for improving crop productivity, resource-use efficiency, and resilience under rapidly intensifying environmental stresses. Unlike conventional agrochemicals, biostimulants function by activating physiological, biochemical, and molecular processes that optimize plant performance without directly supplying nutrients or exerting pesticidal effects. This review comprehensively examines the integrated roles of plant-derived and microbial biostimulants in sustainable agriculture, with particular emphasis on microbial-mediated mechanisms underlying plant stress adaptation and rhizosphere functioning. Plant-derived biostimulants, including seaweed extracts, humic substances, protein hydrolysates, amino acids, and chitosan, enhance nutrient acquisition, root architecture, hormonal regulation, and antioxidant defense systems. More importantly, microbial biostimulants, such as plant growth-promoting rhizobacteria (PGPR), endophytic microorganisms, mycorrhizal fungi, actinomycetes, yeasts, and cyanobacteria, exert multifunctional effects through biological nitrogen fixation, mineral solubilization, phytohormone biosynthesis, volatile signaling, osmolyte accumulation, pathogen suppression, and modulation of stress-responsive genes. These beneficial microorganisms reshape rhizosphere microbial communities, improve nutrient cycling, and enhance plant tolerance to drought, salinity, heat, and heavy metal toxicity. Emerging evidence from genomics, transcriptomics, metabolomics, and microbiome-based investigations has further revealed the molecular networks and signaling pathways governing biostimulant-induced resilience and plant-microbe interactions. Despite their substantial promise, inconsistent field performance, formulation instability, regulatory limitations, and inadequate mechanistic understanding continue to restrict their large-scale adoption. This review highlights recent advances in microbial and plant-derived biostimulants while identifying critical knowledge gaps and future opportunities for precision biostimulant engineering, microbiome manipulation, and climate-resilient crop management. The integration of next generation biostimulant technologies into sustainable agricultural systems may significantly reduce dependence on agrochemicals while improving crop productivity, environmental sustainability, and global food security.

Agriculture↗
PubMed2025

Efficient and versatile rapeseed transformation for new breeding technologies.

Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.

Brassica napus↗
PubMedDate not supplied

Calcium and cancer.

For many years the calcium ion has repeatedly cropped up in research work concerning the transformation of the normal cell to the malignant state. When these results are collected together they indicate that calcium may hold the key to the solution of many of the problems concerning the cancer cell.

Animals↗
PubMed2026

Mammalian growth factors enhance regeneration in transgenic tomato lines.

Genome editing technologies are now available for many crop species, greatly enhancing our ability to investigate gene function and transforming the field of plant transgenesis. However, the capacity to regenerate whole plants from cell culture remains a major limiting factor in many crops. Even in species with regeneration potential, certain genotypes remain recalcitrant. The physiological state of plant cells plays a central role in growth and development and is closely associated with kinase-mediated signaling networks. Notably, several defense-related genes activated during cellular repair processes following transgenesis share significant homology with mammalian defense genes. In this study, we evaluated whether supplementation with three mammalian growth factors could enhance regeneration efficiency in tomato. We selected two cytokines and a pro-inflamatory factor showing homology with plant kinase genes. We compared the percentage of transgenic plants generated through CRISPR-Cas9-mediated mutagenesis of four genes involved in sugar and organic acid metabolism across six tomato lines exhibiting varying regeneration capacities. Over three years of transformation experiments, we demonstrated that the addition of mammalian growth factors during transgenesis significantly improved regeneration frequency, particularly in recalcitrant tomato genotypes. Furthermore, growth factor supplementation not only enhanced transformation efficiency in difficult-to-transform lines but also increased the production of stable secondary lines.

Solanum lycopersicum↗
PubMed2026

Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.

The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.

Oryza↗
PubMed2026

The Rise of Plant Pan-Genomes: From Genome Variation to Predictive Breeding.

Plant pan-genomics is entering a new phase beyond genome variation discovery, requiring a shift from cataloguing genomic diversity toward understanding how variation generates biological function and breeding value. Here, we propose that the future of plant pan-genomics will be shaped by three conceptual transitions. First, structural variation (SV), presence-absence variation (PAV), and haplotype diversity should be interpreted not merely as genomic differences, but as regulatory components that influence gene networks, chromatin organization, and complex traits. Second, the expansion from species-level pan-genomes to genus-level super pan-genomes provides an evolutionary framework for uncovering adaptive genetic modules preserved in wild relatives and overlooked during domestication. Third, integrating pan-genomes with pan-omics, three-dimensional genome analyses, and artificial intelligence will enable the transformation of genomic variation into predictive models for crop improvement. We further propose that the ultimate value of pan-genomes lies not in generating increasingly complete genome collections, but in establishing a mechanistic bridge between genome diversity, biological function, and breeding decisions. This transition will move crop improvement from empirical selection toward rational genome design, where evolutionary diversity can be systematically interpreted, predicted, and engineered.

Journal Article↗
PubMed2024

Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

Solanum lycopersicum↗
PubMed2025

Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

Gene Editing↗
PubMed2026

The emerging impact of CRISPR and gene editing on global crop improvement.

The advent of CRISPR-based genome editing has revolutionized crop improvement, offering unprecedented precision and efficiency in modifying key agronomic traits. This review comprehensively examines the mechanisms, applications, and future potential of CRISPR technology in enhancing global crop production. CRISPR-Cas systems, originally identified as adaptive immune mechanisms in bacteria and archaea, have been repurposed for targeted genome editing in plants. The CRISPR-Cas9 system, in particular, has emerged as a powerful tool for introducing site-specific double-strand breaks, enabling precise genetic modifications. The three-stage process of adaptation, expression, and interference underlies the CRISPR mechanism, with guide RNAs directing Cas endonucleases to specific genomic loci. Advances in CRISPR technology have expanded its applications beyond gene knockouts, encompassing base editing, prime editing, and epigenome editing. These innovations have facilitated the development of crops with enhanced yield, stress tolerance, disease resistance, nutritional content, and post-harvest quality. However, challenges related to off-target effects, regulatory hurdles, ethical concerns, and public acceptance must be addressed to fully harness the potential of CRISPR in agriculture. Integration of CRISPR with other cutting-edge technologies, such as synthetic biology, artificial intelligence, and high-throughput phenotyping, holds immense promise for accelerating crop improvement efforts. As research continues to refine CRISPR tools and expand their applicability across diverse plant species, this transformative technology is poised to play a pivotal role in shaping a sustainable, resilient, and productive global food system for future generations.

Gene Editing↗