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An efficient protocol for Agrobacterium-mediated transformation of tomato cultivars Sandra and Rocky was conducted to examine the possibility of producing transgenic tomato plants cultivars harbouring the nptII gene, conferring kanamycin resistance. To achieve this aim, tomato cotyledon explants were transformed using EHA105 Agrobacterium tumefaciens strain harboring the binary vectors pBI121 which contains Gus gene, and neomycin phosphotransferase II (nptII) as selectable marker gene under the control of a CaMV35S promoter and nopaline synthase (nos) Terminator. Transformant detection was carried out in three distinct ways. First antibiotic selection, Kanamycin at a concentration of100 mgl-1 found to be efficient for this purpose. Second histochemical GUS assay revealed the presence of blue colored zones in a number of shoots and leaves for both in vitro and the greenhouse-grown transgenic plants. Third PCR analysis indicated positive result by showing the fragment for nptII gene in tested transformants, while was absent in non-transgenic control (wild type). On the other hand, the results showed that Sandra cultivar was more efficient for regeneration and subsequently transformation frequency than Rocky cultivar, which record 26.66% of transformation frequency compared with 11.57% in Rocky cultivar.

In this paper we study equivalence classes of binary vectors with regards to their rotation by using an algebraic approach based on the theory of linear feedback shift registers. We state the necessary and sufficient condition for existence of an equivalence class with given cardinality and provide two formulas. The first represents the sharp distribution of cardinalities for given length and Hamming weight of binary vectors and the second enables us to determine the number of different classes with the same cardinality.

BackgroundRice (Oryza sativa) is the main food for half of the world’s population, and is considered the model for molecular biology studies of monocotyledon species. Although the rice genome was completely sequenced about 15 years ago, the function of most rice genes is still unknown.ResultsIn this study, we developed a vector toolkit that contains 42 vectors for transient expression studies in rice protoplasts and stable expression analysis in transgenic rice. These vectors have been successfully used to study protein subcellular localization, protein-protein interaction, gene overexpression, and the CRISPR/Cas9-mediated gene editing. A novel feature of these vectors is that they contain a universal multiple cloning site, which enables more than 99% of the rice coding sequences to be conveniently transferred between vectors.ConclusionsThe versatile vectors represent a highly efficient and high-throughput toolkit for functional analysis of rice genes.

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

Random insertion of T-DNA into the maize genome requires the generation of many transgenic events, resulting in high cost and extensive development time. In contrast, site-specific transgene insertion (SSTI) in highly stable genomic regions emerges as an interesting and more viable alternative, as it allows obtaining elite lines in less time and effort. FLP/FRT and CRISPR/Cas9 homology-directed repair (HDR) strategies, as well as their combinations, are currently the most effective for SSTI in maize. The FLP/FRT system still depends on generating a high number of transgenic events, selecting a recombinant target line (RTL), and co-transforming this RTL with a second T-DNA, since there is no prior information on whether the insertion site is considered stable. In turn, CRISPR/Cas9 HDR requires prior information about the insertion site. From this principle, SSTI is effectively targeted by CRISPR/Cas9 HDR, requiring a smaller number of transgenic events. Furthermore, other strategies have been used for SSTI in animal cells and other plant species, and are very promising for establishment in maize as well. Herein, we highlight the importance of SSTI and the advances made in identifying genomic safe harbors in the maize genome. Furthermore, we emphasize the potential of the FLP/FRT system, CRISPR/Cas9 HDR, and CRISPR-associated recombinases and polymerases. We also offer insights into binary and ternary vector strategies, transgene delivery systems, maize tissue culture, and SSTI event genotyping. Finally, we highlight the importance of rigorous quality control for elite lines containing STTI. Therefore, this study provides insights and trends into SSTI in maize mediated by recombinases and genome editing endonucleases.

The plant kingdom abounds in countless species with potential medical uses. Many of them contain valuable secondary metabolites belonging to different classes and demonstrating anticancer, anti-inflammatory, antioxidant, antimicrobial or antidiabetic properties. Many of these metabolites, e.g., paclitaxel, vinblastine, betulinic acid, chlorogenic acid or ferrulic acid, have potential applications in medicine. Additionally, these compounds have many therapeutic and health-promoting properties. The growing demand for these plant secondary metabolites forces the use of new green biotechnology tools to create new, more productive in vitro transgenic plant cultures. These procedures have yielded many promising results, and transgenic cultures have been found to be safe, efficient and cost-effective sources of valuable secondary metabolites for medicine and industry. This review focuses on the use of various in vitro plant culture systems for the production of secondary metabolites.

Summary In plants, genetic manipulations are commonly performed using Agrobacterium tumefaciens, a plant pathogen whose abilities as a ‘natural genetic engineer’ have been co‐opted for biotechnological applications. In the widely used binary vector systems, Agrobacterium already contains a plasmid with most of the genes needed for virulence and is transformed with a second vector carrying the DNA of interest that will be transferred to the plant. Many widely used binary vectors have not been substantially improved during the past few decades, however, and often have legacy features that are not needed for many contemporary applications, such as cumbersome cloning sites that introduce sequence ‘scars’ between proteins of interest and any fusion protein, including GFP. Here, we introduce the assembly vector (AVEC) plasmids, a series of modular vector backbones that are easily customized using inexpensive DNA assembly cloning methods and that are ideal for testing multiple epitope tags, fluorophores, or non‐coding sequences, such as promoters or 5′/3′ untranslated regions. We show that pAVEC plasmids match or exceed the performance of commonly used binary vectors for transient and transgenic expression of proteins fused to fluorophores and the proximity labelling tool, TurboID. To illustrate the versatility of the pAVEC system, we demonstrate that replacing an herbicide resistance gene with a silencing suppressor gene increases protein expression in cells by an order of magnitude. We anticipate that the pAVEC system will accelerate investigations of plant molecular biology and that the modular, intentional design of these plasmids will be useful to investigate future improvements to binary plasmid design.

The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.

Melia volkensii , a native dryland tree species in Eastern Africa, is extensively grown for its timber that resists termites, as well as for firewood and animal fodder. This study presents the first successful genetic transformation of Melia volkensii mediated by Agrobacterium tumefaciens. A new binary vector enabled the introduction of the reporter gene M24::eGFP resulting in robust gene expression. This breakthrough facilitates future genetic improvement of M. volkensii , including applications of CRISPR-Cas-9 technology, to develop superior varieties for reforestation and sustainable land management in arid regions. Key message A redesigned GFP reporter construct and the use of a phenotyping camera allowed to develop an efficient and robust Agrobacterium mediated transformation protocol for Melia volkensii .

Feature vectors are extracted to represent objects in many classification tasks, such as text classification. Due to the high dimensionality of these raw feature vectors, the classification efficiency and accuracy are reduced. Therefore, reducing the size of feature vectors by selecting the relevant features that better represent the objects is an important aspect in text classification. Feature selection not only reduces the dimensionality of the feature vectors, but also produces more efficient classification models with higher predictive power. In this paper, we propose ARTC, which is an effective feature selection method that is based on the extraction of association rules to classify text documents. The extracted association rules discover the hidden relationships and correlations between the relevant words within the textual documents of a class and a cross different classes. Consequently, each class of documents is represented by a small set of contrasting features that are more effective in text classification. Our experiments show that ARTC outperforms other relevant techniques in terms of classification performance and efficiency.