Agrobacterium has the ability to transfer T-DNA, a part of a tumor-inducing plasmid or Ti-plasmid, into the genome of many plants. A T-DNA binary system is a unique system, which was developed based on this mechanism. This system has been widely used in Agrobacterium-mediated plant transformation. This article provides you with a guide about the T-DNA binary system and some common elements of T-DNA binary vectors.
In this article:
What is a Ti or Ri plasmid?
A Ti or Ri plasmid is a plasmid naturally present inside Agrobacterium. This plasmid is important for successful integration of DNA into the plant genome.
Agrobacterium-mediated plant transformation requires at least three main elements:T-DNA, vir genes, and some additional genes located on the bacterial chromosomes. Both T-DNA and vir genes are present in the Ti or Ri plasmids of wild-type Agrobacterium.
Ti or Ri plasmids are large, low copy number, and hard to isolate. In addition, these plasmids have no multiple cloning sites or MCS. The Ti and Ri plasmids are also unable to replicate in Escherichia coli, typically used for cloning a large piece of DNA on a vector (Lee & Gelvin, 2008).
It is challenging to insert the gene of interest into Ti or Ri plasmids for plant transformation. Therefore, one way to overcome these issues is by using a T-binary system.
What is a T-DNA Binary System?
A T-binary vector system is a method to perform plant transformation by two vectors. The system has two parts: T-binary vector and the vir helper plasmid.
1. The T-binary vector
This vector contains T-DNA repeats from the Ti-plasmids and a gene of interest to be inserted into the plant genome. The T-binary vector has none of the tumor-associated gene sequences and vir genes, so it is much smaller than the size of Ti plasmids.
2. The vir helper plasmid
The second plasmid carries vir genes encoding for Vir proteins, and serves as a helper. Agrobacterium strains can carry a wild-type Ti plasmid or a disarmed Ti plasmid without tumor-related genes, to synthesize Vir proteins. The activity of Vir proteins is essential for integration of the DNA segment into the plant genome by assisting in the cleavage of the T-DNA and the transfer of it from the Agrobacterium cell into the plant cells.
What are Some Features of T-DNA Binary Vectors?
In addition to T-DNA repeats, the T-DNA binary vectors usually contain:
- An origin of replication or ORI for E. coli or OriE: a particular element on the plasmid for starting its replication in E. coli. This component is useful for allowing maintenance of the vector in E. coli.
- An origin of replication or ORI for Agrobacterium or OriA: a particular site on the plasmid for starting its replication in Agrobacterium.
- Multiple cloning sites or MCS: this region containing restriction enzyme sites to allow the insertion of the gene of interest.
- Plant selectable marker: to allow for selection of the transgenic plants.
- Bacterial selectable marker: to allow for selection of the transformed bacteria.
- Promoter: a site to drive transcription of the gene of interest.
- Poly(A) signals: an element containing poly-A, important to produce a protein.
- Reporter: a sequence encoding a particular protein with a specific function for monitoring the recombinant protein, such as β-glucuronidase or GUS, luciferase or LUC, or Green Fluorescent Protein or GFP).
Below is a table containing some features of popular T-DNA binary vectors (Lee & Gelvin, 2008):
After cloning your gene of interest into the T-binary vector, the next step is to transform this vector into Agrobacterium competent cells. For your convenience, all Goldbio’s Agrobacterium competent cells have high transformation efficiency and contain a Ti-plasmid for facilitating efficient T-DNA transfer into the plants.
To learn more about how to choose Agrobacterium competent cells, find GoldBio’s article 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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