Why agrobacterium used for transformation

Weed Res. Murashige T, Skoog F. A revised medium for rapid growth and bio Assays with tobacco tissue cultures. Physiol Plant. Plant Mol Biol. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system.

Bioeng Bugs. Gateway R -compatible plant transformation vectors. Bertani G. Studies on lysogenesis I. J Bacteriol. Agrobacterium tumefaciens-mediated barley transformation. Plant J. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep. Barley transformation using Agrobacterium-mediated techniques. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery, vol.

Jelili OT. Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechnol Mol Biol. Agrobacterium-mediated gene transfer to cereal crop plants: current protocols for barley, wheat, triticale, and maize.

Int J Plant Genomics. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat.

Vitro Cell Dev Biol Plant. Agrobacterium-mediated large-scale transformation of wheat Triticum aestivum L. Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker.

Funct Plant Biol. Transgenic plant regeneration from wheat Triticum aestivum L. Acta Phytophysiol Sin. Agrobacterium-mediated transformation and plant regeneration of Triticum aestivum L. Biol Plant. Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower. Plant Cell Tissue Organ Cult. Agrobacterium-mediated transformation of durum wheat Triticum turgidum L.

Activity of constitutive promoters in various species from the Liliaceae. Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Genet Breed. Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants.

Acta Hortic. Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. The relationship between cognitive failures, psychoneurotic symptoms and sex. Acta Psychiatr Scand. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium.

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. T-DNA binary vectors and systems. Generation of backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Download references. Also, we thank JIC Horticulture and photographic services. Sadiye Hayta, Mark A.

Smedley, Selcen U. You can also search for this author in PubMed Google Scholar. SH developed the method, carried out data assessment and drafted the paper. MAS performed molecular cloning, contributed to the analysis, participated to draft the paper.

SUD and RB contributed the initial experiments. AH and NA participated to optimise the transformation system. WAH supervised and coordinated the project and edited the manuscript. All authors read and approved the final manuscript.

Correspondence to Sadiye Hayta. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions. Hayta, S. An efficient and reproducible Agrobacterium -mediated transformation method for hexaploid wheat Triticum aestivum L.

Plant Methods 15, Download citation. Received : 19 July Accepted : 14 October Published : 26 October Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.

Skip to main content. Search all BMC articles Search. VirA protein can be structurally defined into three domains: the periplasmic or input domain and two transmembrane domains TM1 and TM2. The periplasmic domain is important for monosaccharide detection Chang and Winans, Within the periplasmic domain, adjacent to the TM2 domain is an amphipatic helix, with strong hydrophilic and hydrophobic regions Heath et al. This structure is characteristic for other transmembrane sensor proteins and folds the protein to be simultaneously aligned with the inner membrane and anchored in the membrane Seligman and Manoil, The TM2 is the kinase domain and plays a crucial role in the activation of VirA, phosphorylating itself on a conserved His residue Huang et al.

Monosaccharide detection by VirA is an important amplification system and responds to low levels of phenolic compounds. Recent studies for determination of VirA regions, important for its sensing activity suggested the position, which may be involved on TM1-TM2 interaction. This interaction causes the exposure of the amphipathic helix to small phenolic compounds and suggests a putative model for the VirA-ChvE interaction Doty et al. VirG functions as a transcriptional factor regulating the expression of vir genes when it is phosphorylated by VirA Jin et al.

The C-terminal region is responsible for the DNA binding activity, while the N-terminal is the phosphorylation domain and shows homology with the VirA receiver sensor domain. The activation of vir system also depends on external factors like temperature and pH. The effect of temperature on VirA is suppressed by a mutant form of VirG VirG c , which activates the constitutive expression of the vir genes. However, this mutant cannot confers the virulence capacity at that temperature to Agrobacterium, probably because the folding of other proteins that actively participate in the T-DNA transfer process are also affected at high temperature Fullner and Nester, Generation of T-DNA transfer complex.

The activation of vir genes produces the generation of single-stranded ss molecules representing the copy of the bottom T-DNA strand. These are the only cis acting elements of the T-DNA transfer system. The proteins VirD1 and VirD2 play a key role in this step are, recognizing the T-DNA border sequences and nicking endonuclease activity the bottom strand at each border. The nick sites are assumed as the initiation and termination sites for T-strand recovery.

VirD1 interacts with the region where the ss-T-strand isoriginated. Experiments in vitro evidenced that the presence of VirD1 is essential for the cleavage of supercoiled stranded substrate by VirD2 Zupan and Zambryski, ; Christie et al. The simultaneous restoration of the excised ss-T-strand is evolutionarily related to other bacterial conjugative DNA transfer processes, which includes the generation of the single strand DNA Zupan and Zambryski, ; Christie et al.

Extensive mutation or deletion of the right T-DNA border is followed by almost completely loss of T-DNA transfer capacity, while at the left border results in lower transfer efficiency Hille et al.

Left border may act as a starting site for ssT-strand synthesis but the efficiency is much lower Filichkin and Gelvin, The difference may be a consequence of the presence of an enhancer or "overdrive" sequence next to the right border Peralta and Ream, This enhancer has been found to be specifically recognized by VirC1 protein Toro et al. Deletion of virC operon is followed by attenuation of virulence of the Agrobacterium strains van Haaren at al.

The transferring vehicle to the plant nucleus is a ssT-DNA-protein complex. Is must be translocated to the plant nucleus passing through three membranes, the plant cell wall and cellular spaces. This cooperative association prevents the attack of nucleases and, in addition, extends the ssT-DNA strand reducing the complex diameter to approximately 2 nm, making the translocation through membrane channels easier.

This fact indicates that both proteins presumably play important roles once the complex is in the plant cell mediating the complex uptake to the nucleus Herrera-Estrella et al. The deletion of NLS in one of these proteins reduces, but does not totally inhibit, the ssT-DNA transfer and its integration into plant genome, evidencing the other partner can at least partially assume the function of the absent protein.

It is known that VirE1 is essential for the export of VirE2 to the plant cell, although other specific functions are still uncharacterized Binns et al. Bacterial strains mutated in virE1 , cannot export VirE2 which is accumulated inside the bacterium. Such mutants can be complemented if coinfected with a strain that can export VirE2, indicating that this protein can be exported independently and that the transfer of VirE2 as part of the ss-T-DNA complex is not necessary for the transmission event Sundberg et al.

From these experimental evidences, an alternative model was brought to light for ssT-DNA complex transfer. It is also possible that the process can be performed by one of the proposed alternatives ways according to the infection conditions. Previous researches described the role of 9. VirB are proteins that presents hydrophathy characteristics similar to other membrane-associated proteins Kuldau et al. VirD4 is a transmembrane protein but predominantly located at the cytoplasmatic side of the cytoplasmic membrane Okamoto et al.

Comparative studies showed a high degree of homology between the virB operon and transfer regions of broad host range BHR plasmids in genetic organization, nucleotide sequence and protein function Pohlman et al. Both systems deliver non-self transmissible DNA-protein complex to recipient host cell. The majority of VirB proteins are assembled as a membrane-spanning protein channel involving both membranes Shirasu and Kado, a, b ; Shirasu et al. Except for VirB11, they have multiple periplasmic domains Christie, VirB1 is the only member of VirB proteins found in the extracellular milieu Baron et al.

That could be the case of the VirB2, a protein with deduced extracellular functions. Vir B2 is translated as a 12 kDa proprotein, which is later processed by proteolysis to its mature 7 kDa functional form Jones et al. Vir B11 lacks continuous sequence of hydrophobic residues, formiing periplasmic domains.

Despite these structural characteristics, less than one third of VirB11 constitutes its soluble fraction, while the rest of the protein remains associated with the cytoplasmic membrane Rashkova et al. These characteristics are atypical for this type of protein and evidence the possible dynamic co-existence of different conformational forms in vivo. VirB4 tightly associates with the cytoplasmic membrane Dang and Christie, It contains two putative extracellular domains conferring transmembrane topology to this protein, which presumably allows the ATP-dependent conformational change in the conjugation channel.

The VirB4 synthesis is well correlated with the accumulation and distribution of VirB3. Other protein, VirB7, seems to be crucial for the conformation of the transfer apparatus. VirB7 interacts with VirB9 forming heterodimers and probably higher-order multimeric complexes.

The synthesis of VirB9 and its stable accumulation depends of heterodimer conformation, indicating that VirB9 alone may be unstable and requires the association with VirB7 Anderson et al. In this intermolecular conformation the monomeric subunits are joined by disulfide bridges.

The VirB7-VirB9 heterodimer is assumed to stabilize other Vir proteins during assembly of functional transmembrane channels Fernandez et al. Some of the initial steps of biogenesis of ssT-DNA complex apparatus. Firstly, VirB7 and VirB9 monomers are exported to the membrane and processed.

They interact each other to form covalently cross-linked homo- and heterodimers. Subsequently the VirB7-VirB9 heterodimer is sorted to the outer membrane. The sorting mechanism has not been elucidated Christie, the next step implies the interaction with the other Vir proteins for assembling the transfer channel with the contribution of the transglycosidase VirB1. It is known that VirB2 through VirB11 are essential for DNA transfer, suggesting that these proteins are fundamental component of the transfer apparatus Berger and Christie, , while VirB1 has a lesser contribution to this process.

Two accessory vir operons, present in the octopine Ti-plasmid, are virF and virH. The virF operon encodes for a 23 kDa protein that functions once the T-DNA complex is inside the plant cells via the conjugal channel or independently, as it was assumed for VirE2 export. These Vir proteins are not essential but could enhance the transfer efficiency, detoxifying certain plant compounds that can affect the bacterial growth Kanemoto et al.

If that is the function of VirH proteins, they play a role in the host range specificity of bacterial strain for different plant species.

Integration of T-DNA into plant genome. Inside the plant cell, the ssT-DNA complex is targeted to the nucleus crossing the nuclear membrane. Two Vir proteins have been found to be important in this step: VirD2 and VirE2, which are the most important, and probably VirF, which has a minor contribution to this process Hooykaas and Schilperoort, VirD2 has one functional NLS.

The two NLS of VirE2 have been considered important for the continuos nuclear import of ss-T-DNA complex, probably by keeping both sides of nuclear pores simultaneously open. The nuclear import is probably mediated also by specific NLS-binding proteins, which are present in plant cytoplasm. The final step of T-DNA transfer is its integration into the plant genome.

The mechanism involved in the T-DNA integration has not been characterized. It is considered that the integration occurs by illegitimate recombination Gheysen et al. These homologies are very low and provide jus a minimum specificity for the recombination process by positioning VirD2 for the ligation. This situation activates the repair mechanism of the plant cell and the complementary strand is synthesized using the early inserted T-DNA strand as a template Tinland et al.

VirD2 has an active role in the precise integration on T-strand in the plant chromosome. Jayaram, Agrobacterium -mediated transformation in monocotyledonous plants. Transformation is currently used for genetic manipulation of more than species of at least 35 families, including the major economic crops, vegetables, ornamental, medicinal, fruit, tree and pasture plants Birch, , using Agrobacterium -mediated or direct transformation methods.

The idea that some species cannot accept the integration of foreign DNA in its genome and lack the capacity to be transformed is unacceptable under the increasing number of species that have been transformed.

However, efficient methodologies of Agrobacterium -mediated gene transfer have been established mainly for dicotyledoneous plants Figure 3.

These plantshave been considered to be outside the Agrobacterium host range and other gene-transfer methods were developed for them. To develop these methodologies for a monocotyledoneous plant it is important to take in account the critical aspects in the Agrobacterium tumefaciens- plant interaction, the cellular and tissue culture methodologies developed for that species.

The suitable genetic materials bacterial strains, binary vectors, reporter and marker genes, promoters and molecular biology techniques available in the laboratory, are necessary for selection of the DNA to be introduced. This DNA must be able to be expressed in plants making possible the identification of transformed plants in selectable medium and using molecular biology techniques to test and characterize the transformation events for review Birch, The optimization of Agrobacterium tumefaciens- plant interaction is probably the most important aspect to be considered.

It includes the integrity of the bacterial strain, its correct manipulation and the study of reaction in wounded plant tissue, which may develop in a necrotic process in the wounded tissue or affect the interaction and release of inducers or repressors of Agrobacterium virulence system. The type of explant is also an important fact and it must be suitable for regeneration allowing the recovery of whole transgenic plants.

The establishment of a method for the efficient regeneration of one particular species, is crucial for its transformation. It is recommended to work firstly on the establishment of the optimal conditions for gene transfer through preliminary experiments of transient gene expression using reporter genes Jefferson et al. This opinion is supported by the fact that Agrobacterium -mediated gene transfer is a complex process and many aspects of the mechanisms involved remain unknown.

The expression of opine synthesis coding genes produces opine—the type depends on bacterial strain, an exclusive nutrition for Agrobacterium. In young tissue, swelling is observed from the fourth or fifth day after bacterial inoculation, well-developed in a month, and growing rapidly until it reaches an inch or two in diameter for several months [ 10 ]. The overall mechanism of AMT is summarized in Figure 6. Overall mechanism schematic of AMT.

The wild-type Ti-plasmids are not suitable for being gene vectors because the T-DNA has oncogenes that cause tumor growth in host cells. Construction disarmed Ti-plasmid by deletion of oncogenes, and opine biosynthetic coding gene makes the plasmid non-oncogenic, the 25 bp of each repeat border sequence remaining. Selectable marker genes are inserted into the T-DNA in order to distinguish the transformed cells from normal cells, tandem with experimental transgenes.

Some herbicide resistance markers that are commonly used are phosphinothricin, chlorsulfuron, sulfonamide, and glyphosate. The insertion of bacterial selectable marker, such as trimethoprim, streptomycin, spectinomycin, sulfonamides, bleomycin, hygromycin, kanamycin, neomycin, or gentamicin, evaluates the uptake engineered plasmid to bacterial cell [ 8 ]. Disarmed Ti-plasmid is difficult to be manipulated in vitro due to its large size.

Furthermore, scientist constructs the binary vector, so called because it is designed to be replicate in multiple host E. The binary vector consist of left and right borders, origin of replication for multiple host, selectable marker genes, and gene s of interest.

This engineered plasmid is now used in plant genetic transformation. AMT is a general method for genetic modification in many plant species.

It is because it allows efficient insertion of stable, un-rearranged, single-copy sequences into plant genome. Two critical points for successful transformation were indicated: the use of actively dividing embryonic callus cells derived from the scutella of mature seeds as the starting material and the addition of a phenolic compound, acetosyringone, in the cocultivation steps [ 44 , 45 ].

Moreover, Cheng et al. In general, Agrobacterium -based method was used for transgenic plant. The protocol consists of seven steps, which can be briefly summarized as follows: stage I preparation of sterilize seed or samples and inoculum; stage II explant preparation, infection, and cocultivation with A. The protocol of Agrobacterium -mediated transformation. Immature embryo was a common sample that is used for transformation.

Some experience reported that transformation efficiencies depend on the genotype or variety [ 47 , 48 ]. To obtain the immature embryo, seed is planted in sterile media such as husk, compost, mixed soil, etc. Immature embryo is harvested after pollination, but it depends on the species. On the other hand, callus is also produce from hypocotyl or cotyledon explants. Inoculum is prepared by culture A.

Inoculum should be prepared fresh. In some cases, the growth of A. On the other hand, the A. The embryonic, immature embryo or callus is able to be used as the explants. The explant should be sterilized before the infection or transformation process. Both of the suspension of the embryos and bacteria are transferred to the new plate or empty petri dish. The A.

We can follow some recommendation from several protocols for specific species. Selection is one of the critical factors in the success of transformation.

The process of selection can be occurred after the stage of transformation, regeneration, or on T 0 and T 1 plant. Moreover, antibiotic selection is one of the methods to check the successful transformation. In addition to antibiotic selection, PCR should be used to confirm the presence of the targeted transgene in each transformant at each generation. Regeneration of transformed plants occurred after the proliferation. Trends in plant science, 5 10 , Hwang, H.

The Arabidopsis Book, 15, e A simplified and efficient Agrobacterium tumefaciens electroporation method. Ratjens, S. Frontiers in Plant Science, 9. Nester, E.

Frontiers in Plant Science, 5. Subramoni, S. Agrobacterium tumefaciens responses to plant-derived signaling molecules. Tzfira, T. Agrobacterium : from biology to biotechnology. In this article: What is Agrobacterium - mediated transformation? What is Agrobacterium? How does Agrobacterium infect plants? Why is Agrobacterium used to make transgenic plants? T-binary system 2. Agrobacterium Competent Cells 3. Transformation Efficiency 2. Antibiotic Resistance 3.

There are at least three main components to prepare before performing Agrobacterium -mediated transformation: 1. T-binary system T-binary system is a system commonly used to make transgenic plants.

Referring to figure 3, the following are components of the T-binary vector: T-DNA — shown at the top as a region containing the multiple cloning site MCS MCS or multiple cloning site — to insert a gene of interest Components for selection — the plant selectable marker and bacterial marker Components for replication — origin of replication for Agribacterium OriA and origin of replication for Escherichia coli OriE.

Components for gene expression — these elements include promoter, polyA signals. Component for monitoring the recombinant protein in the transformed plant — a reporter Whereas, the second vector, such as a Vir Helper Plasmid , carries vir genes.

Plants The transformation of plant cells by using Agrobacterium commonly involves incubating the cells or tissues with the bacteria. Transformation Efficiency When performing your experiments, start with Agrobacterium competent cells with high transformation efficiency. Antibiotic Resistance Some strains of Agrobacterium have a particular antibiotic resistance.

Compatibility with the Plants Before starting your plant transformation, find a well-established protocol for your target plant and the Agrobacterium strain compatible with the plant.