Agrobacterium is nature’s genetic engineer. This bacterium has the ability to transfer a part of its DNA into the genome of the plant and uses the plant to provide nutrients for its survival. Based on this remarkable Agrobacterium system, scientists have developed a powerful tool for plant transformation.
This article provides you with a quick review about the Agrobacterium system, why Agrobacterium is useful for plant transformation, and how to choose Agrobacterium competent cells for your research.
In this article:
What is Agrobacterium-mediated transformation?
Agrobacterium-mediated transformation is a process of using Agrobacterium to transfer a gene of interest into the plant cells, generating transgenic plants.
What is Agrobacterium?
Agrobacterium is a soil pathogenic bacterium. This plant pathogen causes crown-gall disease or hairy root disease in infected dicotyledonous plants.
How does Agrobacterium infect plants?
Agrobacterium strains, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes, infect plants and cause the development of large tumor or root hairs in plants.
Fig 1. Agrobacterium infection in the host plants. The Agrobacterium infects a
plant cell. The T-DNA within
Agrobacterium integrates with the plant’s
genome causing the plant to develop crown gall disease (in the case of
or another type of tumor).
Tumor inducing (Ti) or root inducing (Ri) plasmids carried by Agrobacterium play a significant role in this process. A plasmid is a small and circular DNA. Agrobacterium transfers a part of Ti or Ri plasmids, called T-DNA, to plant cells. T-DNA carries a number of genes, important for the survival of Agrobacterium and the bacterial infection in plants.
Fig 2. The Ti plasmid carries a T-DNA region, vir genes and genes encoding opine catabolism. The T-DNA region contains tumor-associated genes, causing abnormal growth in plants by encoding plant growth hormone. It also has opine synthase genes, encoding for enzymes involved in the synthesis of opine.
Among the genes located on the Ti or Ri plasmids, some virulence genes, called vir genes, encode Vir proteins in Agrobacterium.
The activity of Vir proteins promotes the T-DNA integration into the plant genome. Not only Vir proteins, some other proteins encoded by genes in the bacterial chromosomes are also important for this integration.
After detecting plant molecules, Agrobacterium activates its chromosomal genes (chv genes) and vir genes. Several Chv proteins participate in Agrobacterium attachment to the plant cells, whereas Vir proteins assist in the cleavage and the transfer of T-DNA from the bacterial cell into the plant cell.
T-DNA also contains genes encoding plant growth hormones. When expressed in the plant genome, the overproduction of plant growth hormones in plants stimulates the development of tumor or root hairs.
Some genes, located on the T-DNA, encode for enzymes important for the synthesis of unique amino acids, called opines. After T-DNA integration, the large crown galls in the plants provide Agrobacterium with this essential nutrient.
Why is Agrobacterium used to make transgenic plants?
Agrobacterium is a useful tool for plant transformation because it can carry, transfer, and integrate a gene of interest into the plant genome.
In the development of transgenic plants, this system allows plants to stably harbor and pass a particular gene of interest to the next generations relatively quicker than by using the more traditional plant breeding method.
This method is relatively inexpensive and easy to perform. In addition, it provides convenient way to screen and select the transformed plant tissues.
There are at least three main components to prepare before performing Agrobacterium-mediated transformation:
T-binary system is a system commonly used to make transgenic plants. This system contains two vectors. A vector is a plasmid used for cloning, transferring, or expressing the gene of interest.
The first vector is called T-binary vector. It has T-DNA, the gene of interest, and other components needed for selection, replication, and gene expression.
Figure 3: Illustration of the T-binary system showing both vectors. The first vector is the T-binary vector on the left. The second vector, on the right, is the Vir Helper Plasmid.
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. Some strains of Agrobacterium contain either a wild-type Ti plasmid or a disarmed Ti plasmid without tumor-associated genes, as a helper plasmid.
To learn more about T-DNA Binary Vectors, find GoldBio’s article below:
2.Agrobacterium Competent Cells
Agrobacterium competent cells are cells able to take up the T-binary vector containing T-DNA and the gene of interest. The transformed Agrobacterium cells typically thrive at low temperatures and grow much slower than the transformed E. coli cells. These transformed cells are then used for plant transformation.
Fig 4. Transformed Agrobacterium cells are
incubated with plant cells, and therefore infect the plant cells. Afterward,
the plant cells are grown and selected for on a plate. After selection, the
transformed plants are grown.
The transformation of plant cells by using Agrobacterium commonly involves incubating the cells or tissues with the bacteria. The type of plant tissues commonly used is embryonic callus cultures due to the known genotype compared to seedlings, regeneration potential and the stability of the regenerated plants (Ratjens et al., 2018). A callus is a plant tissue, formed from a wound site or cut plant surface.
An additional step after exposing the tissue to Agrobacterium often improves the efficiency of Agrobacterium-mediated gene delivery, including using glass beads or performing sonication (Tzfira & Citovsky, 2007, pg. 76). These methods help weaken the barrier for Agrobacterium to infect the plant cells and transfer DNA.
How to Choose Agrobacterium Competent Cells
When choosing Agrobacterium competent cells, some factors to consider:
When performing your experiments, start with Agrobacterium competent cells with high transformation efficiency. High transformation efficiency is an important feature for Agrobacterium cells because these cells enable to take up T-DNA efficiently.
The most efficient transformation of Agrobacterium is by using electroporation (Kámán-Tóth et al., 2018). To make sure you have this type of cells, choose commercially available cells, such as GoldBio’s Agrobacterium Electrocompetent cells.
Some strains of Agrobacterium have a particular antibiotic resistance. Therefore, avoid using a vector with the same antibiotic marker to carry your gene of interest.
As an example, the EHA101 strain is an Agrobacterium strain resistant to kanamycin. On the other hand, pBIN19 vector uses kanamycin as a selection marker. Therefore, it’s hard to select the cells taking up the vector by using this antibiotic.
Fig 5. Choosing Agrobacterium strain, which carries the same antibiotic resistance with the T-binary vector, causes the growth of cells with the T-binary vector and cells without the vector after transformation.
3.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. Some factors can affect successful plant transformation, including the susceptibility of the plant to Agrobacterium infection, the efficiency of Agrobacterium-mediated gene delivery, and the ability of the plant to express the protein and regenerate whole plants from transformed cells (Tzfira & Citovsky, 2007, pg. 76).
To help you choose and compare the features of GoldBio’s competent cells, learn more from additional information bellow.
Browse GoldBio Agrobacterium competent cells below:
GV3101 Agrobacterium Electrocompetent Cells (Catalog # CC-207)
AGL-1 Agrobacterium Electrocompetent Cells (Catalog # CC-208)
LBA4404 Agrobacterium ElectroCompetent Cells (Catalog # CC-220)
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