Tissue Culture uses tiny pieces from a plant, which we call explants, to produce hundreds, sometimes thousands of plants. Sounds too good to be true, right? What’s the catch?
The catch is that the tissue culture needs to be performed under strict aseptic conditions to avoid contamination. It is relatively simple to maintain an aseptic environment, but what happens when you need to transfer plant tissue under harsh conditions? Keep reading to discover tips and tricks to keep your tissue culture plants contaminant-free, even under the harshest conditions.
Sterile conditions need to be observed at all times, including when the explants are cultured in a nutrient-rich medium. The growing conditions will vary according to each plant species. However, you will always be expected to maintain this sterile environment and avoid contamination - even during divisions and transfers. Once the explants are cultured in the growing medium, you will notice rapid new shoot development. Soon, you will have to divide and transfer the plantlets. This is where it can get tricky - even one bacterial cell or just one spore can compromise your plantlets or the media, and without you even knowing it, the contamination can multiply and cause severe damage.
Transferring Plant Material
Sterilization is key for successfully transferring plant material. Use the following steps to make sure your plant material is kept safe and sound, and always use sterilized equipment and gloves throughout the process.
- You will need to have a sterilized container, PPM™ for sterilizing the plant material, sterile blades and forceps, sterilized paper towels (as a surface to cut on), and tubes that contain the sterile medium. Before you move the tubes into the chamber, you should wipe them down with alcohol (at least 70%) - this includes the capped tubes, the outside surface areas of containers, and any other supplies.
- For extra precaution, spray your gloves with the alcohol solution. After you have sprayed them, rub your hands together so that the alcohol can reach the entire surface area. Ideally, this is done just before you place them into the chamber.
- Once you have your gloves on, avoid touching anything outside of the aseptic chamber.
- Only once all the above precautions have been met should you open the plant material’s container carefully. Pour in the sterilized water so that the container is at 50% capacity. Close the lid and give the container a gentle shake (do this for approximately 3 minutes). This helps to wash and sterilize the explants and remove any excess sterilizing solution. If you have used bleach instead of PPM™, make sure to repeat this process two or three more times.
- Once you have completed the above step, remove the now sterilized plant materials and put them on your prepared petri dish or paper.
- The cutting process will depend on the species that you are propagating.
- After you have prepared your plants, close the cap on the tubes (make sure this is done tightly). After some time, you will notice the new explant will develop a callus, and shoots will eventually develop after it is in the tissue culture media for several weeks.
It helps if your culture is transferred in batches, so if one batch does become contaminated, the damage control is contained in that one batch.
Here are some extra tips for working with your tissue culture so that you can make sure that your plants remain free from harmful contaminants:
- Always keep hair tied back
- Wear a lab coat and always use gloves
- Using a laminar hood flow can dramatically reduce the chance of contamination, especially when passaging cells
- Use ethanol or at least 70% alcohol solution to wipe down surfaces
- Keep all equipment sterilized
- Keep all equipment and materials labeled; this will help to keep you organized and will minimize the chance of contamination
- Once your transfers are complete, write all observations in a journal. This includes the methods used and the results attained.
Various microorganisms can contaminate your plant tissue cultures. While antibiotics are often used, they are not sustainable for long term use as plants can develop genetic mutations, and the antibiotics will eventually be ineffective. PPM™ is a broad-based, superior alternative to antibiotics.
Why you Should Use PPM™
As we have mentioned, PPM™ is a broad-based solution that can be used for sterilization and contamination control. PPM™ is also effective against fungal contaminations, whereas antibiotics are ineffective against fungal growth. PPM™ is a cost-effective alternative to antibiotics, and it is also non-toxic for most plant species.
Do you have any tips that we didn’t mention? Let us know your favorite ways to manage contamination in your tissue culture transfers.