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At least 19 recordsLinked to original sources
PubMed1992

A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.

A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.

Agrobacterium tumefaciens↗
PubMed2026

SpacerScope: binary-vectorized, genome-wide off-target profiling for RNA-guided nucleases without prior candidate-site bias.

The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive sequence-based off-target candidate discovery remains a computational bottleneck, particularly in large and complex genomes. Here we developed SpacerScope, an off-target candidate discovery framework that enables unbiased, genome-wide discovery by leveraging binary vectorization, bitwise filtering, and right-end-anchored alignment. Benchmarking against human CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6142/6142), matching the sensitivity of exhaustive algorithms. Crucially, SpacerScope achieved this maximum candidate recovery while substantially reducing computational overhead. In large-genome evaluations, SpacerScope maintained low peak memory usage of 2.20 GiB and achieved substantial runtime improvements over indel-aware comparator tools, including more than 50-fold speedup relative to Cas-OFFinder 3 (544 s versus 29 185 s). Furthermore, comparative analyses in polyploid species, such as the octoploid strawberry, revealed that SpacerScope identified larger sequence-compatible candidate burdens than standard web-based design platforms. Our results establish SpacerScope as a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes. The source code and program was publicly available at https://github.com/charlesqu666/SpacerScope. Short Abstract CRISPR/Cas sequence-based off-target candidate discovery remains computationally challenging in large, repetitive, and polyploid genomes. Existing tools either miss indel-containing candidate sites or incur prohibitive runtime and memory costs. We developed SpacerScope, a binary-vectorized framework that enables unbiased, genome-wide off-target candidate discovery without pre-selected candidate sites. By integrating bitwise filtering with right-end-anchored alignment, SpacerScope recovered 100% of validated off-target sites in human CIRCLE-seq data while using only 2.20 GiB of memory and achieving more than 10-fold speedup over indel-aware alternatives. Evaluation in plant genomes, including rice and octoploid strawberry, further demonstrated SpacerScope's capacity to identify larger sequence-compatible candidate burdens overlooked by standard tools. SpacerScope thus provides a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes, supporting downstream prioritization.

CRISPR-Cas Systems↗
PubMed1992

New plant binary vectors with selectable markers located proximal to the left T-DNA border.

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.

Agrobacterium tumefaciens↗
PubMed2024

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Genetic Vectors↗
PubMed1992

Construction and use of Agrobacterium tumefaciens binary vectors with A. tumefaciens C58 T-DNA genes.

Five plant morphoregulatory genes were isolated from the Agrobacterium tumefaciens Ti plasmid and binary plasmid vectors for plant transformation with these genes were constructed. All vectors have a similar structure with T-DNA borders, RK2 origin of replication and chimeric kanamycin resistance gene for the selection of transformed plant tissues. Over twenty vectors with single and combined morphoregulatory genes were constructed and their effects after tobacco tissue transformation studied.

Agrobacterium tumefaciens↗
PubMed1992

Factors influencing Agrobacterium-mediated transient expression of gusA in rice.

Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis.

delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.

Bacillus thuringiensis↗
PubMed1992

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer beta-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3' polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Amino Acid Oxidoreductases↗
PubMed1991

Detection of gene regulatory signals in plants revealed by T-DNA-mediated fusions.

A binary vector, pPRF120, was designed to detect T-DNA insertions within transcriptionally active areas of the plant genome. Linked to the right-border repeat, the vector contains a promoterless beta-glucuronidase (GUS) gene which can, upon integration into chromosomes, be activated by cis-acting regulatory elements. The vector also incorporates a chimeric marker gene conferring resistance to kanamycin to ensure recovery of gene fusions regardless of the extent of their tissue-specific or developmentally regulated expression, and to permit analysis of the frequency of plants which express the promoterless reporter. Approximately 1000 transgenic tobacco plants harboring pPRF120 were regenerated. Analysis of 52 individuals indicated that more than 80% contain single, intact copies of the T-DNA, regardless of their ability to express the promoterless GUS gene. Screening of leaf tissue from the 1000 pPRF120 transformants revealed that ca. 5% of the plants contained GUS activity. Fluorogenic and histological GUS assays were used to visualize and quantify tissue- and cell-specific gene expression. The potential usefulness of pPRF120 in comparison to other vectors designed to generate in vivo gene fusions is discussed.

Agrobacterium tumefaciens↗
PubMed1992

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells.

Different factors involved in the early steps of the T-DNA transfer process were studied by using a beta-glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.

Agrobacterium tumefaciens↗
PubMed1991

An alternative approach for gene transfer in trees using wild-type Agrobacterium strains.

Micropropagated shoots of three forest tree species, poplar (Populus tremula x P. alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J. regia), were inoculated each with six different wild-type Agrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with an Agrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carrying neo (kanamycin resistance) and uidA (beta-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to express neo and uidA genes. These results suggest that wild-type Agrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.

Culture Techniques↗
PubMed2026

Targeted expression of Glycine max isoflavone synthase enhances daidzein and genistein content in soybean.

Isoflavonoids are key secondary metabolites in leguminous plants that play essential roles in plant physiology and provide significant health benefits to humans. In the isoflavone biosynthetic pathway, isoflavone synthase (IFS) catalyzes the conversion of naringenin and liquiritigenin into the bioactive isoflavones genistein and daidzein. This study aimed to enhance genistein and daidzein accumulation in soybean seeds through genetic engineering. Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301 containing GmIFS under the control of a seed-specific promoter (Gmβ-conglycinin) was used to transform modified half-seed explants of soybean cv. JS335. Hygromycin-B-resistant plants were regenerated, hardened, and confirmed by histochemical GUS assay. Molecular analysis by PCR validated the presence of the GmIFS transgene, yielding a 700 bp amplicon. Biochemical analysis revealed that seeds of T₀ transgenic plants showed a 1.53-fold increase in total phenolic content and a 3.67-fold increase in flavonoid content compared to non-transformed controls. Antioxidant assays demonstrated significantly higher DPPH radical-scavenging activity and ferric-reducing antioxidant power (FRAP) in GmIFS-overexpressing plants. HPLC analysis further indicated that transgenic seeds accumulated, on average, 4.07-fold higher daidzein and 1.75-fold higher genistein levels relative to control plants. qRT-PCR analysis showed significantly elevated GmIFS expression in immature cotyledons, mature cotyledons, and seeds of transgenic plants. Overall, these results demonstrate that GmIFS overexpression effectively enhances isoflavone production in soybean seeds, highlighting the potential of metabolic engineering of biosynthetic pathway genes to improve nutritional quality.

Glycine max↗
PubMed1979

Using bibliometric analyses of patent literature for predicting the clinical fates of developing drugs.

Certain bibliometric features of the early literatures of developing drugs can be used to predict their ultimate clinical fates. The chronological sequence of publications is expressed as a binary vector with 1 for a patent and 0 for a nonpatent. The decimal equivalents for standardized vector lengths provide scalar values for comparing one drug with another. In order to incorporate concordant patents, fuzzy subsets are employed, with the number of attempts required to achieve transitive closure being the values for comparison. The methods involved are described using minoxidil as an example.

Bibliographies as Topic↗
PubMed1991

Evidence for T-DNA mediated gene targeting to tobacco chloroplasts.

The integration of foreign DNA into plant cells by Agrobacterium mediated transformation is random and normally directed at the nucleus. Here we present evidence that such transformation can be used to introduce foreign genes into higher plant chloroplasts by site-specific homologous recombination. A binary vector was made in vitro that included chloroplast ribosomal DNA (rDNA) within T-DNA borders, and transgenic tobacco plants obtained using this construct were analyzed for the targeted recombination by hybridization, polymerase chain reaction (PCR) and DNA sequencing.

Base Sequence↗
PubMed1992

The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus.

The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.

Amino Acid Sequence↗
PubMed2025

Construction and Segmental Reconstitution of Full-Length Infectious Clones of Milk Vetch Dwarf Virus.

The construction of infectious clones (ICs) is essential for studying viral replication, pathogenesis, and host interactions. Milk vetch dwarf virus (MDV), a nanovirus with a multipartite, single-stranded DNA genome, presents unique challenges for IC development due to its segmented genome organization. To enable functional analysis of its genome, we constructed full-length tandem-dimer-based ICs for all eight MDV genomic segments. Each segment was cloned into a binary vector and co-delivered into Nicotiana benthamiana, Nicotiana tabacum, Vicia faba, and Vigna unguiculata plants via Agrobacterium-mediated inoculation. Systemic infection was successfully reconstituted in all host plants, with PCR-based detection confirming the presence of all viral segments in the infected leaves of nearly all tested plants. Segmental accumulation in infected plants was quantified using qPCR, revealing non-equimolar distribution across hosts. This study establishes the first complete IC system for MDV, enabling reproducible infection, replication analysis, and quantitative segment profiling. It provides a foundational tool for future molecular investigations into MDV replication, host interactions, and viral movement, advancing our understanding of nanovirus biology and transmission dynamics.

Nicotiana↗
PubMed2025

Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Plants, Genetically Modified↗