December 14, 2021
Interested in learning how plant tissue culture works? Or, curious about how Nepenthes have unique requirements within tissue culture? Then, this page is for you!
Before we dive in, a few things to note…
We’ve intentionally kept this article simple to be more beginner friendly. If you’re looking for something more advanced, we’ve recommended additional readings at the bottom of this article.
Additionally, this page uses definitions and frameworks from other posts. We strongly recommend reading the following articles before beginning here:
Tissue culture is a special way of propagating plants. It allows plants to be asexually reproduced at a large scale and low cost.
At the most basic level, tissue culture is cutting a plant into very small pieces, then exposing these pieces to hormones to help them grow.
Before we dive into the tissue culture process, we need to explain two key ideas: sterile environments and tissue culture media.
A sterile environment is a setting where nothing is living except for the desired material (in this instance a plant). This means there are no bacteria, fungi, viruses, or other microbes alive in a sterile environment.
This is why when you see photos of a plant tissue culture lab, all the plants are inside flasks. Each of those flasks is a sterile environment.
By growing plants in a sterile environment, the risk of a plant being harmed by a pathogen is greatly reduced.
Tissue culture media is another core component of tissue culture.
Media is the substance a plant grows on. It’s an all-encompassing term that includes soil in the Earth, a potting mix from your local garden store, or tissue culture media.
Tissue culture media is a unique combination of water, sugar, and plant growth regulators (PGR’s). Plant growth regulator is a scientific way of saying plant hormones.
Think of PGR’s as a scientist’s way of communicating with a plant. If they expose a plant to one PGR, a plant will grow roots. If they expose a plant to a different PGR, the plant will grow new leaves. PGR’s are the “language” scientists use to communicate with plants that makes tissue culture possible. By using different PGR combinations, scientists can tissue culture virtually any plant.
Now that we have an understanding of sterile environments and tissue culture media, let’s dive into the tissue culture process.
Tissue culture is divided into four main stages: Initiation, Multiplication, Rooting, and Weaning. The stages are sequenced as follows:
So, what are each of these stages?
Initiation is when plant material is taken from a non-sterile environment and put into a sterile environment.
This means to introduce a plant into tissue culture, all other living organisms on and in the plant must be removed. This is quite challenging, as this means any bacteria, fungal spores, or parasites have to be removed or killed. Often this requires both a chemical and mechanical cleaning process, such as scrubbing a plant leaf with isopropyl alcohol or soaking it in a bleach solution.
Once a plant has been cleaned and placed onto growth media, the initiation stage is complete.
Loosely speaking, all plants have two areas – roots and shoots. Roots are what grows below the ground (or in the instance of epiphytes, anchors the plant to a tree). Shoots are what grows above the ground. Shoots is a loose term that encompasses leaves, petioles, stems, etc.
If you want the plant to grow shoots, you place it on a media with the appropriate PGR’s. If you want the plant to grow roots, you place it on media with a different set of PGR’s.
Thus, the multiplication stage is growing a plantlet on multiplication media until it is large enough to be divided into tiny pieces. Then, all of those pieces can grow into individual plantlets.
The two following images are both of plantlets in the multiplication stage. The left image is plantlets that have grown to a large size and are ready to be divided. The right image is plantlets that have recently been divided and are now growing again.
These plants haven’t been exposed to rooting media yet, so they don’t have any roots!
If you want the plant to grow roots, you place it on a rooting media. This means the rooting stage is a plantlet on rooting media. The main difference between multiplication media and rooting media is the PGR’s used.
The below images show plants on rooting media.
Once a plant has been multiplied and rooted, it can be removed from the sterile environment and be potted up!
Plants fresh out of a flask generally lack a cuticle, which is the barrier on the outside of each plant that retains moisture. Because of this, tissue culture plants typically need an extra humid environment until they develop their cuticle.
We frequently get asked questions like – why aren’t there more Nepenthes edwardsiana in tissue culture? While the rarity of N. edwardsiana is a partial answer, it’s also due to how difficult Nepenthes are to tissue culture.
It appears that Nepenthes have an endogenous relationship with a bacteria, a fungus, or both. But since tissue culture requires a sterile environment, the challenge is to sterilize the Nepenthes tissue without killing the plant.
To compensate for this, Nepenthes are introduced into tissue culture via seed, before the symbiotic relationships are established. This means that to introduce a new Nepenthes you need:
Yet, this doesn’t cover the challenges that arise during multiplication and rooting. Since each plant prefers a different media recipe and so little research has been done on Nepenthes tissue culture, labs essentially make educated guesses about which media recipes to use. If the plant doesn’t like the media recipes, the plant could die.
To help them get new species introduced into tissue culture, support your favorite Nepenthes lab! It’s an ongoing effort to conserve these wonderful plants we all love.
If you’re interested in learning more about tissue culture, we strongly recommend Plants from Test Tubes.
Are you interested in working in our lab, either professionally or as a college intern? Feel free to contact us at [email protected]. We’re also looking for a smart developer to help us build a great lab management tool! Please contact us at the same email if you’re interested.
As always, if you have any questions feel free to contact us at [email protected] if you have any questions.