How are genes inserted into plants?
I’ve had this question ever since the day I learned of plant biotech. The answer? Gene insertion methods like Agrobacterium tumefaciens!
Genetic engineering is defined as the act of inserting, deleting, or modifying specific genes in an organism’s genome. Agrobacterium is a professional when it comes to gene insertion. So, you can credit many advancements in medicine, agriculture, and biotechnology to tools like Agrobacterium. Right, but…what is Agrobacterium?
This little guy, only 1–2 micrometers in length, is “the single most important tool in agricultural biotechnology” (Thompson et. al., 2020). Although it isn’t the only gene insertion technique, it is the most efficient, effective, and widespread one.
Here, I hope to give a comprehensive introduction to Agrobacterium and other gene insertion methods by exploring these topics in more detail:
- Common gene insertion techniques
- Agrobacterium in nature
- The Ti plasmid
- Process of Agrobacterium-mediated gene insertion
Let’s get to it!
Common Gene Insertion Techniques
Besides using Agrobacterium tumefaciens (“too-muh-fei-shenz”) to edit genes in plants, there are two other common techniques. (If you’re interested, this paper gives a more in-depth comparison of all three methods.)
1. Biolistics
Let’s start off with a bang: methods of biolistics literally shoot DNA bullets into cells using gene guns! This device, powered by gunpowder in the past, is a gun that uses pressurized helium to discharge gold or tungsten coated with DNA onto cells. A small amount of these wounded cells take in the DNA, and though we’re not sure exactly how it happens, scientists have made conjectures (Lacroix and Citovsky, 2020). It’s thought that after the gold bullet causes some DNA breakage, the cells assimilate the foreign DNA into their own genome during repair through the DNA repair pathway.
Biolistics are able to deliver large amounts of DNA into a wide variety of plants, but they are labor-intensive, cause tissue damage, and are very inefficient (the percentage of cells that go through this process and end up with the desired gene is relatively low).
2. Protoplast transformation & electroporation
A protoplast is a plant cell…without the cell wall. But it doesn’t instantly collapse — its contents are held together by the plasma membrane, a layer that surrounds the cytoplasm and acts as a boundary between the cell’s inner and external environments.
The cell wall is removed using enzymes before DNA is introduced using methods such as PEG-mediated transfection (the use of polyethylene glycol to attach DNA to cell membranes) or electroporation. Electroporation is where cells are shot with an electrical pulse that explodes most of them (as in, they could undergo cell lysis, where the membrane ruptures and releases the cell’s contents). The ones that survive are now permeable and can take up the DNA that they were surrounded by.
One of the drawbacks of this technique is that because cells aren’t used to having their skin dissolved, it’s pretty hard for them to recover. Because of this, this technique isn’t suitable for many species.
Aaand a less scientific reason that this method isn’t commonly used is that most scientists kinda hate it. I mean, if you pipette them too hard, they could explode. If you don’t store them at the perfect temperature, they could explode. If their surroundings aren’t at just the right salinity, they could explode. If you look at them wrong, they could explode.
Okay, maybe not that last one. But you get the point.
(They do be pretty, though.)
Agrobacterium tumefaciens: Nature’s Genetic Engineer
Belonging to the family Rhizobiaceae, Agrobacterium tumefaciens is a soil-dwelling plant pathogen.
In its natural habitat (places like agricultural fields, orchards, and gardens that have a lot of organic matter), it infects wounded plants and forms crown gall tumors that act as nutrient-rich environments to support its own growth. To cause its victims to form those tumors, it developed an innate ability to transfer DNA between itself and plants. How, you ask?
Well, that’s where the Ti plasmid comes in!
The Ti Plasmid: a Vector for Gene Transfer
The Ti (tumor-inducing) plasmid is a large, circular DNA molecule (~200 kb) made up of several distinct regions: the origin of replication, T-DNA (Transfer-DNA) region, and virulence genes. Each of these parts plays an important role in the bacterium’s ability to transfer DNA into plant cells and create tumors.
1. Origin of replication
This region is in charge of replicating and maintaining the Ti plasmid within Agrobacterium. During cell division, they initiate its replication process and ensure that Ti plasmid copies are correctly duplicated and segregated.
2. The T-DNA region
Upon attachment to plant tissue, the T-DNA region is injected into the plant genome by a protein group called T4SS (type 4 secretion system). Defined by its left and right borders, the T-DNA consists of auxin & cytokinin genes that promote uncontrolled cell growth and an opine gene that forces infected plant cells to produce nutrients that feed Agrobacterium.
Inside the Agrobacterium, the open catabolism genes break down and utilize the opines as a source of carbon and nitrogen, further boosting its growth.
As brutal as it is for the plants, you’ve gotta appreciate the beauty of this system.
3. Virulence genes
Virulence genes create the proteins involved in the transfer of T-DNA into plant cells. Collectively, they mediate the recognition of wounded plant tissues, the processing of T-DNA, and its transfer into the plant cell nucleus. In addition, virulence proteins make up T4SS, the molecular syringe that injects T-DNA into plants.
As a whole, the Ti plasmid is a brutal and effective tool that is crucial for the survival of Agrobacterium — and biotechnology labs.
And now…the process of Agorbacterium-mediated gene insertion!
Now that we have a basic understanding of how it works, let’s examine how Agrobacterium is used in labs.
There are some restrictions, though: in nature, Agrobacterium primarily infects woody dicots (plants that sprout with two seed leaves and commonly grows wood) and is incompatible with many species outside of that category (but with scientific modifications, their range can greatly increase). Additionally, some plant species have evolved to detect and destroy Agrobacterium. In these situations, other methods like biolistics must be employed.
Anyways, throughout the following steps, we’ll be using Agrobacterium to insert a desired gene (that may code for a trait like drought tolerance, pesticide resistance, bioluminescence, etc.) into plant tissues.
Feel free to refer back to the below schematic as I walk us through the process!
Of the process below, steps 1 & 2 are no longer done today, but certainly were in the 1980s. At that time, this technology was relatively new and people captured Agrobacterium from the wild to use in labs.
Now instead of removing and modifying the Ti plasmid, we use binary vectors, which are much smaller versions of Ti plasmids that include only the T-DNA region and have already been through the first two steps of the below process. Because at some point, it was discovered that you can take the T-DNA region out of the Ti-plasmid, insert any DNA sequence in between its left and right borders, and the T-DNA would still work perfectly. This significant reduction in size made it much easier to duplicate and insert these binary vectors.
1. Isolation of Ti plasmid
If we were to work with wild type Agrobacterium, we must make a few changes to its Ti plasmid so that instead of making tumors, it delivers just our desired trait. To isolate the Ti plasmid, we must lyse (explode) some Agrobacterium, separate its contents, pick out its Ti plasmids, and then purify the DNA.
By the way, companies like Zymo, NEB, and Qiagen have kits — commercially available! — for the extraction of plasmid DNA.
2. Disarming the Ti plasmid
To prevent our target plants from growing scary gall tumors, we must remove or inactivate the genes inside the Ti plasmid that are responsible for causing them (oncogenes like auxin and cytokinin). This is done by either cutting out those genes using restriction enzymes and then binding them back together using DNA ligase (molecular cloning), or introducing mutations into those genes that render them non-functional (site-directed mutagenesis).
Because these methods don’t have 100% success rates, we must identify the Ti plasmids that have successfully undergone oncogene deletion with markers such as genes of antibiotic resistance (those without it/didn’t properly undergo deletion will die without the antibiotic resistance when antibiotics are applied).
3. Insertion of desired genes
Through molecular cloning techniques (such as restriction enzyme digests used to remove oncogenes), the desired genes are added into the T-DNA. Just like before, markers must be added to the Ti plasmids that have successfully been transformed.
As I was learning about this process, a question kept nagging at me: why can’t we just use the same methods that we used to insert genes into Agrobacterium’s Ti plasmid to insert genes into plant cells? Why go through all that extra trouble?
The answer is this: unlike bacterial plasmids, plant DNA — in the form of chromosomes — can’t be removed to be edited directly, and then put back in. The cell would die.
In addition, plant cells have a much more complex and rigid cell wall structure compared to bacterial cells, which presents a significant barrier to the direct insertion of DNA. While plant cell walls are formed by a dense matrix of polysaccharides that provide structural support and protect against the entry of large molecules like DNA, bacterial cell walls lack rigidity, are relatively permeable, and are easily manipulated or disrupted.
But, Agrobacterium has specifically evolved to be able to punch through a plant cell wall’s defenses — better than any man-made tool can.
4. Verification and selection
After the engineered Ti plasmids are reintroduced into Agrobacterium through bacterial transformation, methods (like polymerase chain reaction (PCR), restriction enzyme digestion, or DNA sequencing) are used to verify that the desired genes really are present in our Ti plasmids.
Note: DNA sequencing is the new hot thing in town; places like Primordium Labs can sequence genes and produce a result within a single day! And for only $15!
Then, the selective markers we inserted earlier come of use. In this step, we apply antibiotics like kanamycin (or a different method depending on the type of marker used) to pick out which colonies of Agrobacterium have the desired gene.
5. Transformation of plant cells
Now, our target plant tissues are placed on a Petri dish with a solution containing edited Agrobacterium. All we have to do is sit and wait for Agrobacterium to do its thing: pulling out its T4SS syringe and injecting T-DNA into its unsuspecting victims. After that, the genomes of the plant cells would be permanently changed: they will forever carry and pass down the Agrobacterium’s T-DNA.
6. Removal of Agrobacterium
To prevent its potential interference with subsequent plant growth or analysis, the Agrobacterium must be removed from our plant tissues.
This is usually done through two steps: first, the plant tissues are washed with sterile water, which gets rid of most Agrobacterium. Then, the plant tissues are placed on a medium that kills Agrobacterium but does not affect the plants.
7. Subsequent growth
The story of Agrobacterium has come to an end, and as the plant continues to grow and be transferred to fresh medium or soil, the few remaining Agrobacterium are gradually and naturally eliminated.
But the plants live on! Although they started as little pieces of leaf tissue, they flourish and grow.
How?
Well…
If you’d like to learn more about them (and what’s up with those ruby red plant tissues?), you can check out my blog on plant tissue culture: “What are these crumby, green…blobs?”
Conclusion
Although the method of Agrobacterium-mediated transformation comes with the drawbacks of limited DNA size (generally under 20 kilobases) and varying efficiency based on factors such as plant species, tissue type, and experimental conditions, it is indisputably the one gene insertion method that rules them all. With high efficiency and accuracy, the ability to transform a wide range of plant species, and a reliable mechanism evolved and perfected in nature, Agrobacterium is a tool that has changed the world of plant biotech forever.
Note: Many people are instantly disgusted when they find out that bacteria are used to create GMOs, leading to widespread public opposition…even though countless scientific studies have proved their safety. This could lead to a suppression of biotech that may have led to higher crop yield, more nutritious food, more durable crops, etc. Very sad. (Maybe this’ll be the topic of a blog in the future? Public opinions are always fun to learn about…)
And thanks for staying with me ’til the end!
