Modification Of Plant Genomes Research Articles

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

Plant genome modification technologies and some aspects of their practical use

Aim. To analyze modern methods of plant genome modification on the example of cereals and to evaluate the prospects of their use and practical significance. Methods. A review of available scientific sources and electronic resources containing data on existing and promising methods of genetic modification of cereals was conducted. Results. During the entire time of its development, mankind solved the issue of creating more productive agricultural crops that were adapted to the climatic conditions of existence. For this, domestication and selection based on spontaneous mutations were traditionally used. New technologies of genetic recombination, which have been developed in recent decades, make it possible to change the genome of plants in certain sites determined by the researcher. This has opened up unprecedented opportunities both in fundamental terms and in a whole range of applied aspects for elucidating the functions of specific genes and targeted improvement of the productivity of plants and some of them properties. Conclusions. Some modern new technologies of plant genome modifications are of significant scientific interest and practical importance. The use of these technologies is extremely important in many practical areas, especially for wheat and barley as the most agronomically important cereal crops in the world. Genome modifications agricultural crops require significant attention in terms of their safety and legal regulation of practical use. Information about modern methods of plant genome modification and directions for their use should be a mandatory element in educational courses on modern biotechnology in specialized educational institutions.

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava

BackgroundPlant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava.ResultsIn this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves.ConclusionsOur results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.

Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.

Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton.

CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton (Gossypium hirsutum L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, Agrobacterium rhizogenes-mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase (GhPDS) gene. A. rhizogenes showed the highest hairy root induction (30%) when the bacteria were cultured until OD600 reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM-1, Lumian-21, Zhongmian-49) and the mutations were detected in GhPDS induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in GhPDS were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.

Plant genome modification: from induced mutagenesis to genome editing.

The snowballing growth of scientif ic data obtained using modern techniques of genome editing (GE) calls for their critical evaluation and comparison against previously applied methods such as induced mutagenesis, which was a leading method of genome modif ication for many decades of the past century, and its application has resulted in a huge diversity of cultivars. However, this method was relatively long and included a number of stages from inducing multiple mutations using different mutagenic factors to crossing and selecting the most valuable cultivars for several generations. A new technology of genetic engineering and transgenesis enabled us to radically reduce the time required to obtain a new genetically-modif ied cultivar to one generation and make the modif ication process more effective and targeted. The main drawback of this approach was that an introduced transgene might uncontrollably affect the other genes of a recipient plant, which led to the limitations imposed on transgenesis application in many countries. These limitations have been effectively surmounted thanks to the development of GE techniques allowing for a precise modif ication within a single gene that in many characteristics make it similar to a natural allele (especially when it comes to ribonucleoprotein complexes), which has paved the way for wide application of GE in routine breeding. The paper reviews the main stages of GE development in its application in plants. It provides short descriptions of different GE techniques, including those using protein editors such as zinc-f inger and transcription activator-like effector nucleases (TALEN), and the CRISPR/Cas9 technology. It lists a number of achievements in using GE to produce new cultivars of higher yield that are resistant to unfavorable factors and have good nutritional properties. The review also considers the de novo domestication approach, which allows for faster obtaining of new cultivars from natural varieties. In the conclusion, the future ways of GE development are discussed.

Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana.

There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies.

Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.

CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP )-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.

Advances and outlook of horticultural bioengineering

A Branch of modern biotechnology for creating unique relevant genotypes is bioengineering that harnesses a spectrum of plant genome modification technologies. The study aimed to analyse the current state of the art in genome modification of fruit and berry crops for more significant (vs. premium pure breeding varieties) deviations of norm in the traits and properties of biotic and abiotic resistance, productivity, fruit quality, etc. First horticultural crop transformation studies aimed at developing protocols based on selectable enzyme marker genes of phosphorylationmediated aminoglycoside antibiotics detoxification. Neomycin phosphotransferase nptII constitutes the most common system of transgenic fruit and berry crop selection. In pome crops, the transgenic selection priorities were resistance to scab (Venturia inaequalis (Wint.) Cke), rust (Gymnosporangium juniper-virginianae Schwein.) and bacterial blight (Erwinia amylovora Burrill, Winslow et al.), higher fruit quality, including bright colouring, and reduced enzymatic browning. In stone crops, it was tolerance to plum pox (PPV), papaya ringspot (PRSV) and Prunus necrotic ringspot (PNRSV) viruses. In berry crops — resistance to Sphaerotheca humuli (DC.) Burrill fungus, grey mould (Botrytis cinerea Pers.), root rot (Phytophthora cactorum (Lebert & Cohn) J.Schrot.) and powdery mildew (Oidium tuckeri Berkeley), as well as higher fruit quality. In citruses — resistance to bacterial canker (Xanthomonas citri sub sp.), citrus ulcer (Xanthomonas axonopodis pv citri), greening disease (Huanglongbing (HLB)) and fungi (Trichoderma harzianum Rifai). In tropical crops — resistance to papaya ringspot (PRSV) and banana streak (eBSV) viruses. Unique FT-phenotype transgenic fruit lines are leveraged in the new FasTrack breeding strategy. Nine fruit and berry transgenic crop lines have now been registered worldwide. Transgenic Arctic apples (Golden, Granny, Fuji), plums (Honey Sweet) and papaya (Rainbow, SunUp, Laie Gold) are industry-approved in fresh and processed form. The transgenic list regulated in the Russian Federation does not include fruit or berry crops.

Applications of CPPs in Genome Editing of Plants.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

Advances in Agrobacterium transformation and vector design result in high-frequency targeted gene insertion in maize.

SummaryCRISPR‐Cas is a powerful DNA double‐strand break technology with wide‐ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre‐defined genomic location remain highly challenging. Here, we report an efficient, selectable marker‐free site‐specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium‐mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium‐mediated transformation.

Genome editing in plants using CRISPR type I-D nuclease

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

Data Mining by Pluralistic Approach on CRISPR Gene Editing in Plants.

Genome engineering by site-specific nucleases enables reverse genetics and targeted editing of genomes in an efficacious manner. Contemporary revolutionized progress in targeted-genome engineering technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related RNA-guided endonucleases facilitate coherent interrogation of crop genome function. Evolved as an innate component of the adaptive immune response in bacterial and archaeal systems, CRISPR/Cas system is now identified as a versatile molecular tool that ensures specific and targeted genome modification in plants. Applications of this genome redaction tool-kit include somatic genome editing, rectification of genetic disorders or gene therapy, treatment of infectious diseases, generation of animal models, and crop improvement. We review the utilization of these synthetic nucleases as precision, targeted-genome editing platforms with the inherent potential to accentuate basic science “strengths and shortcomings” of gene function, complement plant breeding techniques for crop improvement, and charter a knowledge base for effective use of editing technology for ever-increasing agricultural demands. Furthermore, the emerging importance of Cpf1, Cas9 nickase, C2c2, as well as other innovative candidates that may prove more effective in driving novel applications in crops are also discussed. The mined data has been prepared as a library and opened for public use at www.lipre.org.

Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants.

Efficient transformation of numerous important crops remains a challenge, due predominantly to our inability to stimulate growth of transgenic cells capable of producing plants. For years, this difficulty has been partially addressed by tissue culture strategies that improve regeneration either through somatic embryogenesis or meristem formation. Identification of genes involved in these developmental processes, designated here as morphogenic genes, provides useful tools in transformation research. In species from eudicots and cereals to gymnosperms, ectopic overexpression of genes involved in either embryo or meristem development has been used to stimulate growth of transgenic plants. However, many of these genes produce pleiotropic deleterious phenotypes. To mitigate this, research has been focusing on ways to take advantage of growth-stimulating morphogenic genes while later restricting or eliminating their expression in the plant. Methods of controlling ectopic overexpression include the use of transient expression, inducible promoters, tissue-specific promoters, and excision of the morphogenic genes. These methods of controlling morphogenic gene expression have been demonstrated in a variety of important crops. Here, we provide a review that highlights how ectopic overexpression of genes involved in morphogenesis has been used to improve transformation efficiencies, which is facilitating transformation of numerous recalcitrant crops. The use of morphogenic genes may help to alleviate one of the bottlenecks currently slowing progress in plant genome modification.

Functional Analysis of Root microRNAs by a Constitutive Overexpression Approach in a Composite Plant System.

Agrobacterium-mediated transformation is a fast and efficient method for genome modification in plants. In this protocol, we apply this technique for the analysis of root microRNA functionality. The induction of hairy roots constitutively overexpressing a given microRNA precursor allows us, in a simple way, to modify the accumulation of specific mature microRNA and analyze the consequence of this alteration on a phenotype of interest. This method generates ready-to-phenotype "composite plants" with untransformed aerial part and microRNA-overexpressing root system, in about 20days.

Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System.

Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.

CRISPR/Cas precision: do we need to worry about off-targeting in plants?

The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.

Challenges in wide implementation of genome editing for crop improvement

Genome editing technologies using customizable sequence specific nucleases (SSNs) enables precise genome modification in plants, which has revolutionized functional genomics research as well as crop improvement. Applicability of SSN-mediated genome editing ranges from targeted gene knockout and single base modification to multiple gene stacking into desired genomic sites. However, there are still considerable challenges in implementing genome editing technologies for practical crop improvement. Here, we briefly discuss the technological challenges, especially those associated with the delivery of SSNs and with homologous recombination-mediated genome editing, and address some promising solutions to overcome the issues.