Analyzing Plant Gene Expression with Transgenic Plants

Agrobacterium
-mediated Transformation

Introduction of
Foreign Genes via
Agrobacterium-
mediated Transformation

Analyzing Gene Expression
With Transgenic Plants

Introduction of Foreign Genes via Agrobacterium-mediated Transformation

Attempts to regenerate plants from A. tumefaciens tumors have never been successful. We now know that the balance of auxins and cytokinins in the galls are such that regeneration would not be possible. Even though it had been determined that T-DNA was integrated into the plant genome, it was clear that the goal of obtaining transformed plants with specific traits would require engineered strains of A. tumefaciens.

The first step in engineering the T-DNA was to remove the phytohormone genes. This solved the problem of non-regeneration because if an explant that is normally regenerable is infected with a non-oncogenic strain of A. tumefaciens then the callus from that explant could be used to obtain regenerated plants. The problem though was how to identify those cells or plants that had been transformed. This was solved by adding a selectable marker to the T-DNA. The most widely used marker is the neomycin phosphotransferase II (NPT II) gene from the bacterial transposable element Tn5. This gene imparts to the recipient cell the capability to grow in the presence of the antibiotic, kanamycin. These cells are thus considered to be kanamycin resistant.

One important point though, is that the NPT II controlling elements are bacterial in nature and if the gene was to be used for plant transformation it would need eukaryotic controlling elements. The first vectors used the Nopaline synthase (NOS) promoter and 3' polyadenylation sequences. The basic structure looks like this:

NOS PROMOTER --- NPT II --- NOS 3' SEQUENCE

Since that time other controlling sequences have been used successfully. One other important element that has been used is the promoter for the 35S RNA from Cauliflower Mosaic Virus. In most systems this promoter is constitutively expressed, as is the NOS promoter, but the CaMV 35S promoter is 30-50 times stronger. This permits higher level of expression of the gene of interest.

Another important step in developing plant transformation vectors was devising methods of putting your gene of interest into an A. tumefaciens T-DNA for transfer to the plant cell. These genes are generally transferred into the T-DNA via homologous recombination. Before this could be done though, a recipient Ti-plasmid had to be designed that was also non-oncogenic. Two approaches were used. One group replaced all the sequences between the TL and TR borders with pBR322 sequences (Jeff Schell's group in Germany) and the other approach was to delete 75% of the T-DNA including those sequences responsible for oncogenicity (Monsanto).

In addition to the engineered recipient T-DNA, an intermediate vector needed to be designed for integration of the genes to be transferred into the T-DNA. Intermediate vectors have three requirements:

selectable markers
- kanamycin resistance
- hygromycin resistance
- bleomycin resistance
scorable markers
- NPT II activity
- opine production
- beta-glucuronidase activity
- chloramphenicol acyl transferase activity
- luciferase activity
cloning site for the gene of interest
- most vectors have only a few sites for integration or a gene sequences; these sites can be situated next to a promoter (such as NOS or CaMV 35S) and 3' polyadenylation signals for constitutive expression or next to a marker gene such as NPT II or CAT for promoter analysis
sequences for homologous recombination
- T-DNA sequences
- pBR 322 sequences

Scorable markers can also be used as reporter genes. These genes can be placed under the control of a specific promoter. If the necessary cell machinery is present, then the promoter will be activated, RNA polymerase will make the mRNA of the reporter gene and it will be translated. To determine if the gene was activated, plant tissue is treated with the appropriate substrate and expression can be monitored. If expression of the reporter gene is detected then the expression pattern of the promoter can be determined.

Steps for Plant Transformation with Agrobacterium

clone your gene or gene fragment into the multiple cloning site of an intermediate vector
introduce your gene into an acceptor A. tumefaciens strain via triparental mating. (reciprocal recombination between the intermediate vector and the T-DNA region of the acceptor plasmid occurs during triparental mating and the gene is now part of the T-DNA region that will be transferred)
co-cultivate the engineered A. tumefaciens strain containing the gene of interest with an explant from which regenerated plants can be obtained
culture the regenerating explant in the presence of a selectable agent and select cells, shoots and rooted-shoots that are resistant to the agent
screen the regenerated plant for the expression of the selectable and scorable markers screen the transgenic plant for the expression of the introduced gene
grow progeny of the transgenic plant and determine the inheritance of the introduced gene

Classes of Transformation Vectors

Cointegrate vectors - the sequences to be transferred to the plant genome reside on the same plasmid as the vir genes
Binary vectors - the sequences to be transferred to the plant genome and the vir region genes reside on different plasmids

Binary vectors offer several advantages to the cointegrate vectors. The primary advantage is that they are much easier to construct since they do not require sequences for cointegration into the Ti-plasmid. Cloning sequences into them is generally easier since you can transform the engineered plasmid into standard E. coli strains. Finally, specialized sequences can be added to them such as cos sites so theoretically large fragments of plant DNA can be cloned into them and the fragment can then be introduced into the recipient A. tumefaciens strain using packaged phage particles.