Agrobacterium
Agrobacterium is a genus of bacteria known for its ability to transfer genetic material into plant cells, leading to the formation of crown gall tumors. This process is used in biotechnology to introduce desired genes into plants for agricultural purposes. Agrobacterium-mediated genetic transformation has been a valuable tool in creating genetically modified crops with improved traits such as pest resistance and increased yield.
7 Key excerpts on "Agrobacterium"
eBook - ePub- S. Umesha(Author)
- 2019(Publication Date)
- CRC Press(Publisher)
...The bacterium can be utilized to obtain functional products using engineered DNA segments of interest. In this process, the desired DNA segment is cloned into the T-DNA region of ‘disarmed’ plasmid, which is then introduced into Agrobacterium and subsequently transferred to plants. From these ‘disarmed’ plasmids, genes responsible for tumour formation are removed. This ensures that the transformed cells are regenerated into fertile plants that transmit the desired DNA fragment to their progeny. Agrobacterium can be used for the transformation of recalcitrant plants, fungi, and human cells under laboratory conditions. Agrobacterium- mediated transformation is regarded as a valuable model system for the study of host–pathogen recognition and delivery of macromolecules to target cells. The interaction of Agrobacterium with plant cells involves the following steps: recognition, expression of virulence (vir) gene, attachment to the host cell, targeting of Vir factors and T-DNA into the host cell, and chromosomal T-DNA integration (Figure 3.1). Expression of vir gene is induced in Agrobacterium after its exposure to phenolic compounds from plants. This chemical recognition, after physical interaction of bacterium and plant cells, induces the formation of bacterial transfer machinery. The whole machinery facilitates the transfer of T-DNA strand into the host plant cell along with a number of Vir factors. After entering the host plant cell, the T-DNA is translocated into the nucleus, which on expression reprogrammes plant cells for a tumour phenotype. 3.4.1 Recognition of Plant Cells as Host by Agrobacterium The presence of Ti plasmid is a selective advantage of Agrobacterium species; some strains lack this plasmid and are able to survive independent of plant host. However, because plant transformation is a complex process and energetically demanding, vir gene expression must be carefully regulated...
eBook - ePub- Kirsi-Marja Oksman-Caldentey, Wolfgang H. Barz, Kirsi-Marja Oksman-Caldentey, Wolfgang H. Barz(Authors)
- 2002(Publication Date)
- CRC Press(Publisher)
...Popular selection systems in current use are listed in Table 2. III. Agrobacterium -MEDIATED TRANSFORMATION A. Agrobacterium tumefaciens: A Natural Gene Transfer System Agrobacterium tumefaciens is a Gram-negative soil bacterium responsible for crown gall disease, a neoplastic disease of many dicotyledonous plants characterized by the appearance of large tumors (galls) on the stems. Virulence is conferred by a large tumor-inducing plasmid (Ti plasmid) containing genes encoding plant hormones (auxins and cytokinins) and enzymes that catalyze the synthesis of amino acid derivatives termed opines (6). The plant hormones are responsible for the deregulated cell proliferation that accompanies crown gall growth, while the opines are secreted by the plant cells and used by the bacteria as food. These genes are contained on a specific region of the Ti plasmid, the T-DNA (transfer DNA), so called because it is transferred to the plant nuclear genome under the control of vir (virulence) genes carried elsewhere on the Ti plasmid (15). It is this natural gene transfer mechanism that is exploited for plant transformation. B. Development of Ti-Plasmid Vectors The earliest indication that T-DNA could be used as a plant transformation vector was the demonstration that DNA from an Escherichia coli plasmid (the Tn7 transposon) could be stably transferred to the plant genome by first incorporating it into the T-DNA (16). However, transgenic plants could not be recovered from the transformed cells, either by regeneration or by grafting onto normal plants, because the hormones encoded by the T-DNA oncogenes caused unregulated and disorganized callus growth (16). In rare cases, shoots were derived from such callus tissue, and analysis showed that much of the T-DNA (including the oncogenes) had been deleted from the genome (17)...
eBook - ePub- Firdos Alam Khan(Author)
- 2018(Publication Date)
- CRC Press(Publisher)
...This restriction can be overcome by using the methods of genetic engineering, which in principle allow introducing valuable traits coded for by specific genes of any organism (other plants, bacteria, fungi, animals, viruses) into the genome of any plant. The first gene transfer experiments with plants took place in the early 1980s. Normally, transgenes are inserted into the nuclear genome of a plant cell. Recently, it has become possible to introduce genes into the genome of chloroplasts and other plastids (small organelles of plant cells that possess a separate genome). Transgenic plants have been obtained using Agrobacterium -mediated DNA transfer and direct DNA transfer, the latter including methods such as particle bombardment, electroporation, and polyethylene glycol permeabilization. The majority of plants has been transformed using Agrobacterium -mediated transformation. 6.2.3.6 Agrobacterium-mediated gene transfer The Agrobacterium -mediated technique involves the natural gene transfer system in the bacterial plant pathogens of the genus Agrobacterium. In nature, Agrobacterium tumefaciens and Agrobacterium rhizogenes are the causative agents of the crown gall and the hairy root diseases, respectively. The utility of Agrobacterium as a gene transfer system was first recognized when it was demonstrated that these plant diseases were actually produced as a result of the transfer and integration of genes from the bacteria into the genome of the plant. Both Agrobacterium species carry a large plasmid (small circular DNA molecule) called Ti in A. tumefaciens and Ri in A. rhizogenes. A segment of this plasmid designated T-(for transfer) DNA is transmitted by this organism into individual plant cells, usually within wounded tissue. The T-DNA segment penetrates the plant cell nucleus and integrates randomly into the genome where it is stably incorporated and inherited like any other plant gene in a predictable, dominant Mendelian fashion...
eBook - ePub- K. G. Mukerji, K.L. Garg(Authors)
- 2020(Publication Date)
- CRC Press(Publisher)
...Thus, there are many sites available for ingress of the pathogen. Following infection the plant cells transform into autonomously proliferating tumor cells. The resultant unregulated cell division gives rise to clearly visible and unsightly galls. The young galls are pale, soft, and smooth, but on aging enlarge, darken and, particularly on woody plants, become hard and deeply fissured. Unlike the majority of plant pathogenic bacteria, A. tumefaciens has a uniquely vast host range among dicotyledonous plants — over 600 species in 93 families are reported to be susceptible. 15 However, the host range of some strains of the pathogen, e.g., those attacking grapevine, is very limited. 37 B. The Causal Bacteria The four species of Agrobacterium generally recognized are distinguished from each other on the basis of their pathogenicity. They are A. tumefaciens, A. rhizogenes, and A. rubi that incite crown gall, hairy root, and cane gall diseases, respectively, and the closely related but nonpathogenic species A. radiobacter. 38 However, since 1970 the unsatisfactory nature of this division of the agrobacteria has become abundantly clear from standard biochemical and physiological tests, studies involving numerical taxonomy, DNA/DNA hybridization studies, and electrophoretograms of soluble proteins. The complexities and current thinking on the relationships of the agrobacteria in the light of these varied studies are admirably reviewed by Kersters and De Ley. 38 The conclusions from all these approaches are that there are three main groups (biovars 1, 2, and 3) within the genus Agrobacterium that differ both genetically and phenotypically, but do not correspond to the above species names based on pathogenic behavior...
eBook - ePubPlant Tissue Culture
Techniques and Experiments
- Roberta H. Smith(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
...Chapter 14 Agrobacterium -Mediated Transformation of Plants Jungeun Kim Park, Sunghun Park, Qingyu Wu and Stuart Sprague Kansas State University Chapter Outline Petunia or Tobacco Leaf Disk Sequence Preculture Explant Preparation Cocultivation with Agrobacterium Shoot Transformation and Elimination of Agrobacterium Antibiotic Stocks Procedure Rooting and Pretesting for Transformed Shoots Procedure Petunia Shoot Apex Sequence Seed Germination Explant Preparation Shoot Induction Explant Preparation Shoot Apex Transformation Rooting Observations Tobacco Leaf Infiltration Sequence Grow Agrobacterium Culture Agrobacterium Harvest Agrobacterium Procedure Infiltration Tobacco Leaves There are three major approaches to inserting foreign genes into plants. These include direct DNA uptake by protoplasts by either electroporation or PEG-induced uptake, biolistics, or Agrobacterium- mediated gene transfer. Protoplast uptake of foreign DNA has been described by Cocking (1960). This technology has resulted in transgenic corn and rice (Datta et al., 1992 ; Rhodes et al., 1988 ; Ren & Zhao, 2008 ; Zhang et al., 2011). A problem with the application of this technology is lack of routine cell culture methods to obtain plants from protoplasts of many important crop species. Biolistics, or microprojectile bombardment, is another exciting technique. This method uses accelerated microcarriers (gold or tungsten particles) to penetrate and deliver DNA into the cell (Klein et al., 1988). This particle delivery system has resulted in gene expression in many different plants and plant parts...
eBook - ePub- Julian Coleman, David Evans, Anne Kearns(Authors)
- 2020(Publication Date)
- Taylor & Francis(Publisher)
...Chapter 13 Bacterial culture in the plant cell culture laboratory 1. Introduction The production of transgenic plants requires methods for identifying, isolating, replicating and inserting DNA into the plant genome. All these steps may involve the use of bacteria and bacterial culture (especially of E. coli and Agrobacterium tumefaciens) is routine in the plant tissue culture laboratory undertaking plant transformation. Thus while the culture techniques required are different from those for plant and cell culture, a basic description will be given here. A vector (which may be either bacterial or viral) is needed to introduce a gene into a foreign genome. The vector system most commonly used for plant transformations is based on the tumour-inducing plasmid of A. tumefaciens. Types of transformation vectors are described in detail in Chapter 4. Prior to insertion into a transformation vector, the isolated gene must be integrated into a cloning vector — a bacterial plasmid which can be multiplied in a bacterium like E. coli that is easy to handle in the laboratory. E. coli and A. tumefaciens are available as modified strains that minimize risk to the environment. E. coli cells treated with calcium chloride will take up plasmids from the culture medium and replicate them. They can be grown to logarithmic phase, incubated in calcium chloride and snap-frozen as competent cells — ready to take up a plasmid when required (Protocol 13.1). Fragments of DNA can also be packaged into bacteriophage λ, which can be used to transform the bacteria. Agrobacterium rhizogenes is also used as a transformation system for plant cell cultures, where it produces ‘hairy’ roots — roots with abundant root hairs. These have been used in tissue culture as they are effectively immortal and produce abundant and rapidly growing root cultures (Chapter 10). Techniques for the culture of A. rhizogenes are therefore also included with those for E...
eBook - ePub- L.C. Fowke, F. Constabel(Authors)
- 2018(Publication Date)
- CRC Press(Publisher)
...One of the problems is that although various genes exist in multiple copies or multiple gene families, many others do not and are thus such tiny parts of the large amount of DNA in a plant cell. Many complex traits that are of agricultural importance, such as yield and resistance to biotic and abiotic factors, are polygenic traits which are neither well identified nor biochemically characterized. With a suitable plant vector and the appropriate DNA, methods are available for introduction of this DNA into the genome of plant cells, where it is expressed in RNA which can be translated into stable, active proteins. The expression of such genes can be achieved by making the vector construction in such a way that the gene of interest is controlled by the regulatory sequences (promotor region) of a plasmid gene. This can be the marker gene of the vector. III. The Ti Plasmid Of the transformation systems which exists in nature the Ti plasmid present in the soil-bacterium Agrobacterium tumefaciens meets the conditions described here and might be a good candidate as a vector. A. tumefaciens is the bacterium that causes crown gall disease on dicotyledonous plants. Crown gall is characterized by unlimited plant cell proliferation (gall formation). Crown gall tumor tissue differes from nontransformed tissue by its ability to grow autonomously on synthetic media in the absence of hormones, i.e., auxins and cytokinins. Moreover, tumor-specific compounds, called opines, are produced by these cells as a direct result of the genetic transformation by the bacterium. Opines are unusual amino acid derivatives which accumulate in the plant cells. They appear not to have any known useful function in the plant cells and are not responsible for the tumorous state of the cells. However, opines can be useful to agrobacteria, since they can serve as a source of carbon and nitrogen, while some of the opines also induce conjugation between the bacteria. The Ti plasmid of A...
