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"Genetic transformation"
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Developing a flexible, high‐efficiency Agrobacterium‐mediated sorghum transformation system with broad application
Summary Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi‐arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium‐mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free ‘quality events’ ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84‐7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9‐mediated gene knockouts in Tx430.
Journal Article
Advanced textbook on gene transfer, gene therapy and genetic pharmacology : principles, delivery and pharmacological and biomedical applications of nucleotide-based therapies
This advanced textbook provides a clear and comprehensive description of the field of gene delivery, gene therapy, and genetic pharmacology, with descriptions of the main gene transfer vectors and a set of selected therapeutic applications, along with safety considerations.
Book
Technological Development and Application of Plant Genetic Transformation
Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.
Journal Article
CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation
Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as
Drosophila
and mouse. However, gene targeting in higher plants, including the most widely used model plant
Arabidopsis thaliana
, remains challenging. Here we report a sequential transformation method for gene targeting in
Arabidopsis
. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific
DD45
gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the
Arabidopsis
genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the
Arabidopsis
genome.
Efficient gene targeting in higher plants remains challenging. Here, the authors develop a sequential transformation method for CRISPR/Cas9-mediated gene targeting in
Arabidopsis
and demonstrate its functionality at five genomic sites in two endogenous loci.
Journal Article
The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation
Although great progress has been achieved regarding wheat genetic transformation technology in the past decade1–3, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency4–11. In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration. The use of TaWOX5 in improvement of transformation efficiency also showed promising results in Triticum monococcum, triticale, rye, barley and maize.Over-expressing TaWOX5 substantially increases the transformation efficiencies of wheat and other cereals, including barley and maize, with reduced genotype dependency, and transformed transgenic plants can readily be screened using a visible phenotype.
Journal Article
CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective
Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.
Journal Article
Plant genetic transformation: achievements, current status and future prospects
Summary Regeneration represents a fundamental biological process wherein an organism's tissues or organs repair and replace themselves following damage or environmental stress. In plant systems, injured tree branches can regenerate adventitious buds and develop new crowns through propagation techniques like cuttings and canopy pruning, while transgenic plants emerge via tissue culture in genetic engineering processes intimately connected to plant regeneration mechanisms. The advancement of plant regeneration technology is critical for addressing complex and dynamic climate challenges, ultimately ensuring global agricultural sustainability. This review comprehensively synthesizes the latest genetic transformation technologies, including transformation systems across woody, herbaceous and algal species, organellar genetic modifications, crucial regeneration factors facilitating Agrobacterium‐mediated transformations, the intricate hormonal networks regulating plant regeneration, comparative analyses of transient transformation approaches and marker gene dynamics throughout transformation processes. Ultimately, the review offers novel perspectives on current transformation bottlenecks and proposes future research trajectories.
Journal Article
Nanoparticle-Mediated Delivery towards Advancing Plant Genetic Engineering
Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.
Plant biotechnology is key to ensuring food and energy security; however, biomolecule delivery and progeny regeneration continue to be key challenges in plant genetic engineering.
Conventional biomolecule delivery methods in plants have critical drawbacks, such as low efficiency, narrow species range, limited cargo types, and tissue damage.
Advances in nanotechnology have created opportunities to overcome limitations in conventional methods: nanoparticles are promising for species-independent passive delivery of DNA, RNA, and proteins.
The advent of nuclease-based genome editing (e.g., CRISPR-Cas9) has ushered in a new era of precise genetic engineering that, among other impacts, has enabled the development of genetically engineered crops without harsh regulatory restrictions.
The potential of nanoparticles to overcome limitations in conventional delivery makes them excellent candidates for delivery of nuclease-based genome editing cargo, thus making nanoparticle delivery a critical technology for the advancement of plant genetic engineering.
Journal Article
Methods for genetic transformation of filamentous fungi
Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation,
Agrobacterium
-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.
Journal Article