You may have heard of a technique now commonly being used in plant science: CRISPR/Cas9. But what is this? and why is it causing such a buzz in the science world?
The world of the Science PhD student is shrouded in mystery. What sorts of things do we actually do? How do we spend our days? What experiments do we do?
A few weeks ago I wrote a post about stable plant transformation, which is used for the long-term research of genes, and for long-term production of a trait/compound. This post is going to focus on another plant transformation technique: Transient transformation.
What is transient transformation?
Transient transformation is a mechanism to introduce or silence genes temporarily in plants in order to make specific proteins. This method is very versatile, efficient and quick to do compared to traditional stable plant transformation.
How do you do transient transformation?
Like with stable transformation, the naturally occuring soil bacteria: Agrobacterium tumefaciens is used. A gene of interest is introduced into agrobacterium as outlined here. The agrobacterium containing the desired gene is then grown in liquid culture until a high density of bacteria is reached. This suspension of agrobacterium is then either directly introduced into plant leaves via injection, or into whole plants via vacuum infiltration.
One of the most widely used methods of agrobacterium infiltration is direct injection into plant leaves. For this the agrobacterium solution is put into a syringe with no needle. The tip of the syringe is placed against the leaf, and using a slight pressure, injected into the leaf. The suspension is taken up into the air spaces of the leaf.
Through this method, several different strains of agrobacterium can be injected into different areas of plant leaves at the same time- allowing multiple comparisons of different gene combinations to be studied at the same time.
Click here for a video of agro-infiltration into a tobacco leaf.
This allows deep penetration of the agrobacterium into plant tissues, and can be used to infiltrate entire plants. Normally young plants will be infiltrated by this mechanism. What is particularly good about this mechanism is that a plantlet infiltrated with agrobacterium will continue to grow and show any altered physiological changes, however will not pass any of these changes to its offspring- the change to gene expression is temporary.
For vacuum infiltration the plant is submerged into a solution of agrobacterium, before a vacuum is applied. Under vacuum conditions air is forced out of the intracellular air spaces in the leaves of the plant via the stomata. When the vacuum is then reversed, the change in pressure means the agrobacterium solution is taken up via the stomata into the plant tissue.
Why we use Transient expression systems:
- Transient expression is a much faster method than stable transformation
- Products of gene activity such as recombinant proteins, are made days after initial agrobacterium infiltration.
- It can easily be scaled up for commercial uses
- Co-infiltration of agrobacterium with different genes can be used to produce recombinant proteins
- Can be used for the targeted silencing of genes (supression of gene activity)
Example: commercial use- Vaccine production
The plant Nicotiana benthamiana is a member of the tobacco species- and is ideally suited for use with the transient expression system for commercial production of antibodies and vaccines: as it is extremely quick to grow, and can be used for agrobacterium infiltration after just one week.
Using the transient expression system, virus-like particles (VLPs)- particles designed to look exactly like the polio virus, without the virus-causing element, were produced in the leaves of N. benthamiana. The Virus like particles were extracted from the leaves, and used to make a vaccine which was shown to successfully prevent polio virus in animal studies.
The great thing about this system, is that a large number of plants can be infiltrated with agrobacterium at once, and will actively begin producing its product after just a few days. It is thought that this technology may be able to be used for the production of a wide variety of vaccines, and due to the rapid and cost-effective mechanism, you could potentially go from having no vaccine to a vaccine just 4 weeks after identification of a viral strain. This would be a massive improvement on the timescale of current vaccines- for example a vaccine for flu currently takes a few months to generate. This technology still needs some development, but watch this space!
For more information about Transient expression: (It was very hard to find articles in the public domain which show more about transient transformation- if you know of any, please leave a comment and I will add them to the list below)
In depth science research paper: http://www.sciencedirect.com/science/article/pii/S1369526614000193 (you may need specific access for this)
https://www.jove.com/video/50521/efficient-agroinfiltration-plants-for-high-level-transient-expression (again access may be required)
More about the use of plants for production of polio vaccine: https://www.jic.ac.uk/news-and-events/news/2017/08/plant-produced-polio-vaccines-could-help-eradicate-age-old-disease/
Feauture image source: wikimedia
You may have heard plant scientists saying they used “plant transformation” to express a gene within a target plant. But what exactly does this mean?Continue reading “What is plant transformation: Stable transformation”