What are these crumbly, green…blobs?
An introduction to plant tissue culture
Plants can be genetically edited for a variety of reasons, both practical and aesthetic. Crops have been modified to be resistant to diseases and to grow year-round; pothos to purify air; petunias to be bioluminescent!
But, a full-grown plant has billions upon billions of cells, and genetically modifying a plant means changing the DNA of every one of them. Accomplishing this feat requires these crumbly green blobs, plant callus. They are the equivalent of stem cells in animals, and a single one can regenerate an entire plant on its own, making specialized structures with identical genetic code. This process of using calli to create thoroughly edited plants is called tissue culture.
Tissue Culture
Like some other organisms, plants have the power of pluripotency, the ability to create — from a single undifferentiated cell — different types of specialized cells that can regenerate an entire plant. Methods of tissue culture harness this power by using hormones to convert portions made of specialized cells (leaf, stem, etc.) into pluripotent cells and then prompting that cell to recreate a whole plant.
Let’s explore these aspects of tissue culture in more detail:
- History
- Steps
- Applications & Importance
- Challenges & Considerations
- Future Directions
History
Tissue culture, the growing of small pieces of a plant (explants) in a laboratory setting, was first explored in the 1950s. In 1962, a huge milestone was achieved: the Murashige and Skoog (MS) medium.
Widely used even today, the medium created by Murashige and Skoog provides a balanced combination of nutrients, vitamins, and plant growth regulators, all mixed in a clear agar gel. In their research paper, they stated that the mixture was created specifically for tobacco plants (Nicotiana benthamiana, not Nicotiana tabacum which is used for smoking), but although it still works for many other species, Nicotiana benthamiana is now one of the most commonly used plants for experimental tissue culture, and MS the most commonly used medium.
Steps
There are a few different techniques of tissue culture, some of which are meristem, suspension, embryo, protoplast, and callus culture, which is what I decided to research. Below is a general overview of the steps required to do callus tissue culture, and you can see this document by Sebastian S. Cocioba for a more detailed guide.
1. Prepare explant tissue
The first step in regenerating a complete plant from a portion of the plant is to prepare that small portion of the plant, also called the explant. This tissue can be any part of a plant: leaf, stem, root, bud, or flower (different plants have different types of explant tissue that work best for them.)
Preferably, this tissue comes from lab-grown plants that have always been kept in a sterile environment so that it is free from bacteria and fungal spores inside and out. If this isn’t possible, you will be working with a much higher risk of contamination that no amount of alcohol blasting will completely eliminate. But, it’s always a great idea to sterilize all tools and materials using an autoclave…or a pressure cooker, if, like me, you don’t have access to a fancy lab.
The below picture and all that follow are from an experiment I came across where a gene called RUBY which produces a vivid red betalin (He et al., 2020) — the same one that gives beets their color — was inserted into Nicotiana benthamiana. Callus tissue culture and the gene insertion technique of Agrobacterium (more on that in my blog: “But HOW are genes inserted into plants?”) were used in this process.
2. Prepare tissue culture medium
The tissue culture medium is one of the most important parts of this experiment. There are many different recipes for many different species of plants and types of experiments, but one of the most commonly used mediums is — you guessed it — the Murashige and Skoog (MS) medium! It can be bought online from places like Phytotech Labs, which is a great site to buy all sorts of reasonably-priced equipment and chemicals for plant experiments.
The MS medium contains vitamins and hormones like auxins and cytokinins (more on them later) that are crucial for callus formation and plant regeneration. But, because the MS medium comes in the form of powder and must be supplemented with other nutrients, it must be mixed with water, table sugar (sucrose for nutrition), baking soda (for pH adjustment), agar (for solidification), and the growth regulators — warning: large science words — Benzylaminopurine (BAP) and Napthaleneacetic acid (NAA) to make it into the nutritious gel that is necessary for the next steps.
3. Place explant tissue onto medium to prompt callus formation
As the nutrients in the gel keep the explants alive, the hormones in the medium will prompt the explant tissue to revert back to its pluripotent state. Then, these pluripotent cells will rapidly divide (something that most specialized plant cells can’t do), eventually forming the shapeless, crumbly blobs we call calli.
A key player in this process are auxins. Auxins are a class of plant hormones that regulate many parts of a plant’s behavior and growth process. They take part in basically everything, and plants can’t live without them.
In the RUBY tobacco experiment, Agrobacterium that carried a set of genes called RUBY were mixed with the explant tissue, tasked with the job of infecting the plant tissue with RUBY. (How, you ask? Well, I’ve made a blog about exactly that!) Because most of the Agrobacterium will be unsuccessful in inserting the genetic material, only some of the calli will express that gene. As shown below, these are the ones to grow red tobacco plants.
But…in other gene editing experiments where the desired change doesn’t affect appearance, how would you know which calli carried the inserted gene?
It turns out that the foreign gene would usually contain a sequence that protects an organism from a certain herbicide or other chemical. That chemical would then be applied to all plants to eliminate the ones without the desired gene. Brutal, but effective.
4. Encourage regeneration from callus with cytokinin
Now, it's time for regeneration. Cytokinin is a class of plant hormones that promote growth in developing parts like roots and shoots. They also encourage the calli to start forming specialized structures, restoring order to the chaotic world of calli. They could be thought of as the ‘opposites’ of auxins, which act as triggers for specialized cells to return to an undifferentiated state.
From here, shoots are placed in a medium that allows for rooting (usually an agar gel), so they can then develop into large, complete plants.
After the plant is completely regenerated and roots have started forming, it can be moved into soil for further development. In this experiment, the transgenic tobacco grew stunning red roots because of its modification!
Depending on factors like plant species, explant type, and medium composition, this entire process can take anywhere from 3–8 months, or more. Although it may seem like a painstakingly long time, the work is well worth it — with tissue culture, the possibilities are endless!
Challenges & Considerations
There are two serious challenges around tissue culture.
The first bottleneck is the fact that most plants have a really hard time going through the entire procedure of tissue culture via callus culture. Many of them can make it to the callus stage just fine, but growing back into an organized structure requires a combination of unique genetic, physiological, and environmental factors. For example, the MS medium was a breakthrough: it was the perfect combination of nutrients and hormones that fit the needs of the tobacco plant. This limits the potential of many plants, but fortunately, we have already cracked the code for a good number of species and continue to discover more.
The second challenge is sterility. Fungi and bacteria love agar.
Just like how your rich, nutrient-dense medium boosts callus development, it is also a feast for the countless fungal spores floating freely in the air…and just one will ruin it all. In addition, explant tissue that wasn’t grown and germinated in a sterile environment is likely contaminated inside and out, and the insides can’t be bleached — or the plant will die. Sterility is the main reason — and a common one — for why it is difficult to do these experiments at home. However, it is certainly possible…a makeshift sterile environment could be created in containers like jars or Tupperware if thoroughly cleansed, and a pressure cooker could be your autoclave. After all, anyone can do science, and we all deserve to understand it!
Applications & Importance
The field of tissue culture has two traits that make it an invaluable tool in plant biotech.
First, complete plants can be regenerated from small pieces of plant tissue or even individual cells. In gene editing, only a small number of cells can be edited at once, so it would be impossible to directly edit entire plants. After editing only small pieces of explant tissue (as demonstrated in the red tobacco experiment ) the process of tissue culture allows for the selection and production of complete plants with 100% edited cells.
Second, micropropagation results in the mass (and relatively quick) production of plants with the exact same genes as each other. Micropropagation is the process of taking cuttings from a plant and continuing its growth from already-developed structures (without making cells pluripotent). This contrasts tissue culture, which pertains to making specialized cells pluripotent (where genes are then inserted), and then back into specialized structures. Micropropagating plants after tissue culturing them is necessary for the wide distribution of genetically edited products like enhanced crops, exotic houseplants, and other biotechnologies, so it is most commonly used in industry whereas tissue culture is frequently used in biotechnology labs.
In these two ways, the field of tissue culture has already made possible the exciting technologies of plants that produce more nutritious food, generate more clean air, remove more carbon from the atmosphere, and survive hotter or more challenging environments where water and nutrition are scarce. It has also given us a glimpse into a magical future: one with air-purifying houseplants and glowing petunias!
With new studies and experiments being conducted all the time, the future of plant biotech is more exciting than ever. To learn more about tissue culture and how it can be done at home, I recommend the book Plants From Test Tubes: An Introduction to Micropropagation. The wonders of plant biotech are endless, and glowing houseplants are just a taste of what’s to come.
