How can you prepare sterile explants?
If you have been reading about plant tissue culture, then by now you know how important sterile conditions are for a successful in vitro culture practice. Also, you might be wondering about how to start this sterile process? What exactly you need in order to prepare sterile explants? Where exactly you need to be careful? Well, then you have come to the right space. This article is for you!
In the last article "How to select and prepare stock plants?" we have discussed Stage 0 of plant tissue culture, where you select the stock plants and maintain them in suitable conditions for an efficient end result. These stock/mother plants can provide you with disease-free explants in order to produce a large number of plants.
In this article, we go one step further and talk briefly about Stage 1 of plant tissue culture. The most important thing in this stage is to make sure that the chosen explants are as sterile as possible. Hence we call this stage "the establishment of aseptic cultures". The good thing is that there are several standard protocols that you can use to clean and sterilize the explants. You can work with different protocols for different sizes as well as different types of explants. It is also important to note that these protocols also depend on the plant species you are considering. Overall, the main purpose of this stage is to eradicate external contaminants (bacteria, viruses, fungi, etc.).
Let us dive into some details for this part of tissue culture process:
Firstly, you need to clean the explants after extracting them from the stock plants. You can decide on which type of cleaning protocol to use, based on how "contaminated" your explant is. You also need to consider the sensitivity of the chosen explant to cleaning agents/disinfectants. A too strong cleaning agent will kill your explant, while a too weak agent will not do the job.
You can begin the cleaning process by cleaning the explants with running tap water, so as to remove any soil particles or dead plant tissues. After that, use bleach to clean the explants. In most cases, any household bleaches will do the trick. However, as each brand of bleaches might have a different concentration so you need to make sure to dilute your bleach first. You can also use it along with a series of different disinfectants like ethyl alcohol, etc. depending on the requirement.
You need to keep in mind that, during this process, these agents are not for eliminating internal contaminants.
It is pretty simple to make a bleach solution! Let's take an example here. You can make 1% solution (or a 1:100 solution) by simply mixing 1 ml of bleach with 99 ml of sterile water. And there you go, this will make a solution of 100 ml for your explants disinfection!
When the explants are quite "dirty", it will be helpful to mix a few drops of a surfactant/wetting agent in your bleach concentration.
Did you know about surfactants? Let me try to explain it a bit! So surfactants are compounds that help you to reduce the surface tension between a solid surface and a liquid. In simple terms, surfactants will improve the efficiency of your disinfection process significantly. For you they will work as wetting agents in disinfection treatments.
One of the common wetting agents is Polysorbate 20, popularly known as Tween 20. You might have heard this name before! What wetting agents do, is that it will reduce surface tension to make sure that your bleach concentration will come in complete contact with your explant. It's a simple equation to keep in mind: more contact surface = better sterilization.
I have seen some laboratories use liquid dish soap as a wetting agent. However, for such alternatives, you might run a risk of damaging your explant tissues.
There are quite a lot of disinfectants available in the market. Some examples would be 70% ethyl alcohol, 5%-10% calcium hypochlorite, 3% hydrogen peroxide and so on. However, among these, the most common disinfectant is ethyl alcohol. It is like the staple food of disinfectants, used by almost every plant tissue culture laboratory.
Last but not least is how long should you keep your explants in your cleaning solution. Intuitionally, a solution with higher disinfectant concentration equals less contacting time. Generally, a highly concentrated solution requires a contact time from 15 to 30 minutes. While for lower concentrated solutions, the contact time can vary from one to a couple of hours. There's no fix formula, because each plant species is different. You will have to experiment to find out what works best for your operation.
Apart from clean explants, you also need to have clean hoods (laminar flows), other equipment, culture vessels and every object that you are going to use in plant tissue culture. So while working in a sterile environment you should also make sure that every surface including laminar flow/biosafety cabinets is properly cleaned with 70% alcohol or a household detergent.
I cannot stress enough the importance of laminar flow cabinets. They are there for you to safely make sterile transfers. For example, if you want to transfer a disinfected explant to a culture vessel with culture media or for making subsequent transfers. These flow cabinets come with HEPA filters ('High-Efficiency Particulate Air filter') which gently streams filtered air through the transfer hood to "push" dirty air out of the working space. These filters are very fragile and you should be careful while handling them so as to avoid touching or damaging them.
We hope you got a glimpse of some general, yet important practices to follow in order to maintain a clean and safe environment for culturing plants in any kind of laboratory. For more informational posts on different aspects of plant tissue culture, keep checking this space!
By Nancy Bhatia | 20-April-2021
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