Agrobacterium-mediated Plant Transformation Research Articles

BpWOX11 promotes adventitious root formation in Betula pendula

Adventitious root formation is a key step in vegetative propagation via cuttings. It is crucial for establishing birch plantations and preserve birch varieties. Although previous studies have highlighted role of WOX11 in controlling adventitious root formation, no such study has been conducted in birch. Understanding the mechanism of adventitious root formation is essential for improvement of rooting or survival rate using stem cuttings in birch. In this study, we cloned BpWOX11 and produced BpWOX11 overexpression (OE) transgenic lines using the Agrobacterium-mediated plant transformation. OE lines exhibited early initiated adventitious root formation, leading to increase the rooting rate of stem cuttings plants. RNA sequencing analysis revealed that OE lines induced the gene expression related to expansin and cell division pathway, as well as defense and stress response genes. These may be important factors for the BpWOX11 gene to promote adventitious root formation in birch cuttings. The results of this study will help to further understand the molecular mechanisms controlling the formation of adventitious roots in birch.

Agrobacterial Transformation Enhancement by Improved Competent Cell Preparation and Optimized Electroporation.

The introduction of plasmids into Agrobacterium cells is one of the key steps in the Agrobacterium-mediated transformation of plants for gene editing applications. Depending on chromosomal background, some Agrobacterium strains exhibit a very low transformation efficiency, which results in a low number of colonies for subsequent screening and thus limits the potential for automated high-throughput transformation processes, especially with low copy or large plasmids. This study demonstrates improvements of transformation frequency by modifying the competent cell preparation process and optimizing electroporation parameters for two Agrobacterium strains. The competent cell preparation process was modified by prolonging bacterial growth in the log phase and optimizing the endpoint cell density for cell harvest which resulted in a significant cell yield increase and transformation frequency improvement. Optimization of electroporation by fine-tuning the parameters not only resulted in a 30-fold transformation frequency increase but also revealed a strain-dependent requirement for field strength and electric pulse length. To further improve transformation of a recalcitrant strain, different concentrations of dimethyl sulfoxide (DMSO) in recovery medium were examined. The study revealed an important role of DMSO in transformed cell recovery, with 5% DMSO resulting in the highest transformation frequency. The significant improvements in Agrobacterium transformation frequency addressed a critical bottleneck towards establishing a high throughput process.

Genomic consequences associated with Agrobacterium-mediated transformation of plants.

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Mutation in the Agrobacterium hisI gene enhances transient expression in pepper

Agrobacterium-mediated plant transformation is widely used in plant genetic engineering. However, its efficiency is limited by plant immunity against Agrobacterium. Chili pepper (Capsicum annuum L.) is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation. In this work, Agrobacterium was found to induce a strong immune response in pepper, which might be the reason for T-DNA being difficult to express in pepper. An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves. Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death, presumably due to reduced bacterial growth. However, mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response. The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.

Multiplicity of the Agrobacterium Infection of Nicotiana benthamiana for Transient DNA Delivery.

Biological DNA transfer into plant cells mediated by Agrobacterium represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single versus multiple transfection events per cell. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Here, we analyze the Poisson probability distribution of the T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/Nicotiana benthamiana. These data delineate the relationship between an individual Agrobacterium strain infiltration OD600, plant cell perimeter, and leaf age, as well as plant cell coinfection rates. Our analysis establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf at infiltration OD600 above ∼0.2 for individual strains. In contrast, single-strain T-DNA delivery can be achieved at low strain infiltration OD600: at OD600 0.02, we observe that ∼40% of plant cells are infected, with 80% of those infected cells containing T-DNA product from just a single strain. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

Transgenic tobacco plants expressing a chimeric antimicrobial protein show disease resistance to plant diseases

Plant-pathogen interactions are universal in nature and jeopardise plant health and ultimately global food security. Plant innate immunity can be enhanced by expressing antimicrobial proteins that interfere with the life cycle of plant pathogens. To this end, a recombinant protein comprising a chitinase chitin-binding domain (CBD) from rice and the Phyllomedusa bicolor Dermaseptin B1 (DrsB1) antimicrobial peptide was designed to target fungal cell wall component, chitin. Agrobacterium-mediated transformation of tobacco plants led to the transgenic plants exhibiting significantly resistance to fungal and bacterial plant pathogens. Fungal colonization as well as appearance of fungal disease symptoms delayed for one-week, taking a crucial opportunity from pathogens to establish infections at appropriate time and opportune environmental condition. Furthermore, results of in vitro analysis showed that total protein from transgenic plants significantly hindered growth and development of selected plant pathogens. Predictably, the CBD-DrsB1 recombinant protein had a stronger antimicrobial effect against fungal than bacterial and oomycete pathogens, suggesting that the CBD interferes with fungal cell wall component, chitin oligomers. Scanning electron micrograph (SEM) revealed that CBD-DrsB1 indeed interferes with fungal pathogens by damaging the overall cell wall integrity. Inoculating in vitro transgenic lines with Fusarium oxysporum, Alternaria solani and Pythium sp showed that plants remained green and vigorous and survived up to 51, 56 and 16 days post inoculation (dpi), respectively, whereas non-transgenic plants collapsed completely a few days post inoculation. The results of this study demonstrate that CBD-DrsB1 over-expression limits fungal and bacterial infections, suggesting both in vivo antibacterial activity and possible biotechnological application to engineer crop plants resistant to destructive plant pathogens.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

An efficient and reproducible Agrobacterium-mediated genetic transformation method for the ornamental monocotyledonous plant Ornithogalum dubium Houtt

Ornithogalum is a genus from the Hyacinthaceae (Asparagaceae) family that comprises about 200 species with remarkable white, yellow, or orange flowers that display exceptional vase life. These properties have made it a popular cut flower and pot plant. Forward genetics approaches may be advantageous to generate novel phenotypes, but the Agrobacterium-mediated transformation of plants from this genus remains challenging. Here, a stable and efficient Agrobacterium-mediated transformation system was established for O. dubium. We found that the timing of transformation with respect to light exposure of the tissue affected transformation rates more than other tested parameters. In the transgenic plants obtained, T-DNA integrations were confirmed by polymerase chain reactions and positive plants were established in the greenhouse and displayed weak transgene expression. This study exposed an efficient platform for gene function research and germplasm improvement in O. dubium plants. The present protocol is now available for the development of novel improved O. dubium varieties.

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

BackgroundAgrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner.ResultsBased on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation.ConclusionsOur study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

Selection of the optimal concentration of indole-3-butyric acid (IBA) in the nutrient medium for effective micropropagation of Populus × berolinensis

The study describes the features of the effective micropropagation of Populus berolinensis using indolyl-3-butyric acid (IBA). Internode sections without axillary buds are the most frequently used type of explant for Agrobacterium-mediated genetic transformation of woody plants. The application of growth regulators, such as indolyl-3-butyric acid (IBA), allows to increase the length of the internodes and thereby solve the problem of the availability of a sufficient amount of material for genetic transformation. Our results showed that 0.15 mg L-1 of IBA in the nutrient medium is the most suitable concentration for the effective micropropagation of Berlin poplar. Shoots rooted or regenerated from axillary buds in case of replanting of the rooted stump at medium supplemented with IBA in mentioned concentration, have expanded internodes in comparison with the control group of plants and may be successfully used as a good source of internode sections in Agrobacterium-mediated plant transformation.

A New Approach for Controlling Agrobacterium tumefaciens Post Transformation Using Lytic Bacteriophage.

Overgrowth of Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control Agrobacterium growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28-30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of A. tumefaciens post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated A. tumefaciens (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of A. tumefaciens subsequent to transformation.

FRET-FLIM to Determine Protein Interactions and Membrane Topology of Enzyme Complexes.

Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer with fluorescence lifetime imaging microscopy (FRET-FLIM) is an advanced imaging methodology that allows for the quantitative detection of protein-protein interactions. In this method, proteins of interest for interaction studies are fused to different fluorophores such as enhanced green fluorescent protein (eGFP; donor molecule) and monomeric red fluorescent protein (mRFP; acceptor molecule). Energy transfer between the two fluorophore groups can only occur efficiently when the proteins of interest are in close physical proximity, around ≤10 nm, and therefore are most likely interacting. FRET-FLIM measures the decrease in excited-state lifetime of the donor fluorophore (eGFP) with and without the presence of the acceptor (mRFP) and can therefore give information on protein-protein interactions and the membrane topology of the tested protein. Here we describe the production of fluorescent protein fusions for FRET-FLIM analysis in tobacco leaf epidermal cells using Agrobacterium-mediated plant transformation and a FRET-FLIM data acquisition and analysis protocol in plant cells. These protocols are applicable and can be adapted for both membrane and soluble proteins in different cellular localizations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein expression in tobacco leaf cells via transient Agrobacterium-mediated plant transformation Basic Protocol 2: FRET-FLIM data acquisition and analysis.

The concept of an agroinfiltration kit for recombinant protein production for educational and commercial use-A journey through a forest of regulatory and legal implications.

Recombinant expression using Agrobacterium-mediated transient transformation (ATT) of plants has developed into a robust and versatile method to rapidly produce proteins. The capability of plants to efficiently synthesize even homo- and hetero-multimeric complex folded proteins featuring disulfide bonds and other post-translational modifications such as N-linked glycosylation makes them superior to most of the established microbial, especially prokaryotic expression hosts. Compared to production in mammalian cell cultures, ATT requires lower skills, simple technical equipment and cheaper media components. Taken together these features make the method optimally suited for R&D applications involving the development and engineering of recombinant proteins for various purposes ranging from vaccine candidates, therapeutic proteins, towards enzymes for different pharmaceutical and technical applications. Despite these advantages the technology is currently not being used outside the community of plant research. The design and realization of a kit containing all the information, instructions and ideally also the material required to perform recombinant protein production using ATT in an educational or commercial context was one of the objectives of the EU-funded Horizon 2020 project Pharma-Factory. While it is pretty straightforward to assemble a comprehensive instruction manual describing the procedure, the clarification of regulatory and legal aspects associated with the provision, dissemination and use of the different materials and organisms required to perform ATT is a complex matter. In this article, we describe the initial concept of an ATT kit for educational as well as research and development (R&D) purposes and the specific regulatory and legal implications associated with the various kit components. We cover aspects including intellectual property rights, freedom-to-operate (FTO), safety regulations for distributing genetically-modified organisms (GMOs), as well as export and import regulations. Our analysis reveals that important components of the ATT kit are freely available for research purposes but not or only with considerable effort for commercial use and distribution. We conclude with a number of considerations and requirements that need to be met in order to successfully disseminate such a kit in the future.

Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation

Tumor-inducing (Ti) plasmid is the requisite for Agrobacterium-mediated plant transformation. Over decades, continuous efforts have been made to improve the efficiency of Agrobacterium-mediated plant transformation and most of them focused on the binary vector system. A binary vector system comprises of a binary vector of which transferred DNA (T-DNA) resided on and a Ti plasmid to carry those essential virulence genes. In this study, we constructed a miniaturized helper Ti plasmid, designated as pYL102, with the aim to enhance the overall Agrobacterium-mediated transformation rate. The size of pYL102 was reduced to ~60% of the original plasmid pCAMBIA5105. Subsequently, pYL102 was coupled with the broad host range (BHR) bacterial expression vector, pYL101C, of which the key regulatory virulence gene, virG-N54D, was cloned in and expressed under the control of a strong constitutive PINTc promoter. To test the functionality of the constructed vector system, A. tumefaciens C58C1 carrying pYL102, pYL101C::virG-N54D and the transformation vector pGWB2::e35S-sfGFP was used to transform Nicotiana benthamiana leaves by agroinfiltration. Green fluorescence was observed in spots infiltrated with Agrobacterium carrying the test plasmids. The fluorescence intensity from the test agroinfiltrated leaves was significantly higher than those of the mock-infiltrated leaves (p<0.01), indicating the vector system can be used for plant transformation.

Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties to Agrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineering Agrobacterium tumefaciens to express a type III secretion system (T3SS) from Pseudomonas syringae and individually deliver the P. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineered Agrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineered A. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.

Analysis of T-DNA integration events in transgenic rice

Agrobacterium-mediated plant transformation has been widely used for introducing transgene(s) into a plant genome and plant breeding. However, our understanding of T-DNA integration into rice genome remains limited relative to that in the model dicot Arabidopsis. To better elucidate the T-DNA integration into the rice genome, we investigated extensively the T-DNA ends and their flanking rice genomic sequences from two transgenic rice plants carrying Cowpea Trypsin Inhibitor (CpTI)-derived gene Signal-CpTI-KDEL (SCK) and Bacillus thuringiensis (BT) gene, respectively, by TAIL-PCR method. Analysis of the junction sequences between the T-DNA ends and rice genome DNA indicated that there were three joining patterns of microhomology, filler DNA sequences, and exact joining, and both the T-DNA ends tend to adopt identical manner to join the rice genome. After T-DNA integration, there were several variations of rice genomic sequences, including small deletions at the integration sites, superfluous DNA inserted between T-DNA and genome, and translocation of genomic DNA in the flanking regions. The translocation block could be from a noncontiguous region in the same chromosome or different chromosomes at the integration sites, and the originating position of the translocated block resulted in comparable deletion based on a cut/paste mechanism rather than a replication mechanism. Our study may lead to a better understand of T-DNA integration mechanism and facilitate functional genomic studies and further crop improvement.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods

The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

BackgroundTrachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg’s B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. ResultsAccording to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. ConclusionsIn this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Identification and Validation of Genetic Variations in Transgenic Chinese Cabbage Plants (Brassica rapa ssp. pekinensis) by Next-Generation Sequencing.

Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the ‘CT001’ reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.