-
EcoSal Plus Dec 2022In the late 1950s, a number of laboratories took up the study of plasmids once the discovery was made that extrachromosomal antibiotic resistance (R) factors are the... (Review)
Review
In the late 1950s, a number of laboratories took up the study of plasmids once the discovery was made that extrachromosomal antibiotic resistance (R) factors are the responsible agents for the transmissibility of multiple antibiotic resistance among the enterobacteria. The use of incompatibility for the classification of plasmids is now widespread. It seems clear now on the basis of the limited studies to date that the number of incompatibility groups of plasmids will likely be extremely large when one includes plasmids obtained from bacteria that are normal inhabitants of poorly studied natural environments. The presence of both linear chromosomes and linear plasmids is now established for several species. One of the more fascinating developments in plasmid biology was the discovery of linear plasmids in the 1980s. A remarkable feature of the Ti plasmids of Agrobacterium tumefaciens is the presence of two DNA transfer systems. A definitive demonstration that plasmids consisted of duplex DNA came from interspecies conjugal transfer of plasmids followed by separation of plasmid DNA from chromosomal DNA by equilibrium buoyant density centrifugation. The formation of channels for DNA movement and the actual steps involved in DNA transport offer many opportunities for the discovery of proteins with novel activities and for establishing fundamentally new concepts of macromolecular interactions between DNA and specific proteins, membranes, and the peptidoglycan matrix.
Topics: Plasmids; Agrobacterium tumefaciens; Plant Tumor-Inducing Plasmids; Bacteria; DNA, Bacterial
PubMed: 35373578
DOI: 10.1128/ecosalplus.esp-0028-2021 -
Nature Protocols Jan 2023There 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... (Review)
Review
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.
Topics: Agrobacterium; Nicotiana; Plants, Genetically Modified; Coculture Techniques; Gene Editing; Genome, Plant; Soil; CRISPR-Cas Systems; Transformation, Genetic
PubMed: 36253612
DOI: 10.1038/s41596-022-00749-9 -
Plant Communications Apr 2024Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated...
Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocols and low efficiency of current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of the regenerative activity-dependent in planta injection delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues. Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues. The method was successfully used for transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (Ipomoea pes-caprae). Compared with traditional transformation methods, RAPID has a much higher transformation efficiency and shorter duration, and it does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.
Topics: Plants, Genetically Modified; Agrobacterium tumefaciens; Ipomoea batatas
PubMed: 38243598
DOI: 10.1016/j.xplc.2024.100822 -
Plant Biotechnology Journal Oct 2021Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is...
Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.
Topics: Agrobacterium; CRISPR-Cas Systems; Cannabis; Gene Editing; Mutagenesis; Plants, Genetically Modified; Transformation, Genetic
PubMed: 33960612
DOI: 10.1111/pbi.13611 -
Metal Ions in Life Sciences Mar 2020Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to...
Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ββα architecture consisting in a β-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a βββαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.
Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Humans; Proteins; Zinc; Zinc Fingers
PubMed: 32851833
DOI: 10.1515/9783110589757-018 -
Plant Biotechnology Journal Jul 2024Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be...
Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.
Topics: Gene Editing; Transformation, Genetic; Agrobacterium; Plants, Genetically Modified; CRISPR-Cas Systems; Plant Leaves; Kalanchoe; Gene Transfer Techniques
PubMed: 38425137
DOI: 10.1111/pbi.14318 -
Nature Plants Jun 2020An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are...
An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.
Topics: Agrobacterium tumefaciens; Gene Editing; Plants, Genetically Modified; RNA Viruses; RNA, Viral; Nicotiana
PubMed: 32483329
DOI: 10.1038/s41477-020-0670-y -
Plant Cell Reports Jun 2024A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and...
A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.
Topics: Glycine max; Plant Leaves; Plants, Genetically Modified; Agrobacterium; Gene Expression Regulation, Plant; Nicotiana; Genetic Vectors
PubMed: 38837057
DOI: 10.1007/s00299-024-03245-4 -
Plant Cell Reports Oct 2021This review summarizes the recent advances in legume genetic transformation and provides an insight into the critical factors that play a major role in the process. It... (Review)
Review
This review summarizes the recent advances in legume genetic transformation and provides an insight into the critical factors that play a major role in the process. It also sheds light on some of the potential areas which may ameliorate the transformation of legumes. Legumes are an important group of dicotyledonous plants, highly enriched in proteins and minerals. Majority of the legume plants are cultivated in the arid and semi-arid parts of the world, and hence said to be climate resilient. They have the capability of atmospheric nitrogen fixation and thus play a vital role in the ecological sphere. However, the worldwide production of legumes is somehow not up to the mark and the yields are greatly affected by various biotic and abiotic stress factors. Genetic engineering strategies have emerged as a core of plant biology and remarkably facilitate the crop improvement programmes. A significant progress has been made towards the optimization of efficient transformation system for legume plants over the years but this group is still underutilized in comparison to other crops. Among the variety of available DNA delivery systems, Agrobacterium-mediated and particle bombardment have been primarily deployed for optimization and trait improvement. However, recalcitrance and genotype-dependence are some of the major bottlenecks for successful transformation. In this context, the present review summarizes the advances taken place in the area of legume transformation and provides an insight into the present scenario. The challenges and future possibilities for yield improvement have also been discussed.
Topics: Agrobacterium; Crops, Agricultural; Culture Media; Fabaceae; Genetic Engineering; Genetic Markers; Genotype; Plant Breeding; Plants, Genetically Modified; Transformation, Genetic
PubMed: 34230986
DOI: 10.1007/s00299-021-02749-7 -
International Journal of Molecular... Aug 2021species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of... (Review)
Review
species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.
Topics: Agrobacterium; Chromosomes, Plant; DNA Repair; DNA, Bacterial; DNA, Plant; Plants; Transformation, Genetic
PubMed: 34445162
DOI: 10.3390/ijms22168458