17 Jan 2023

Surface Sterilization Techniques to Prepare Plant Tissues For Culture

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Introduction

The explant is the tissue or plant part you introduce in the culture media for the regeneration of the plant. Therefore, it’s primary to sterilize it well to remove all the dirt, bacteria, or infections stuck on the surface of the explant. It ensures that the resulting plant will be free from any diseases.

The success of tissue culture is based on three main factors:

• To develop contamination-free cultures.
• Induction of plantlet in vitro.
• Transfer of in vitro regenerated plantlets to field conditions.

Thus, it’s necessary to carefully perform each step for higher yield!

So far many surface sterilization techniques have been developed to clean explants. However, the required type, concentration and time of exposure of the disinfectants differ for different plant and plant parts. In this blog, we cover how the sterilization methods are used and a general procedure to sterilize explants. Further we also talk about how you can use PPM (Plant Preservative Mixture—a broad spectrum formulation to eliminate all kinds of contamination) for the purpose.

Types of Explant Sterilization Techniques

Here’re some chemicals most frequently used in labs to sterilize plant explants:

• Sodium hypochlorite: It is also known as bleach. Hypochlorite is very effective in killing bacteria even at a micromolar level. Sodium hypochlorite is one of the most commonly used explant sterilizing chemicals. To use bleach in tissue culture, it is diluted to 10-20%, which results in 0.5-1.0% bleach in the final solution. Each type of explant requires a different amount of time to sterilize. Most of the time, however, 10-20 minutes will suffice. However, the chemical is quite aggressive, which makes calcium hypochlorite a more preferable sterilant.  
• Mercuric chloride: The chemical is highly toxic and that’s why it’s rarely used in labs. When sterilizing the explant using this chemical, it’s essential to rinse it many times using sterile distilled water. There shouldn’t be any traces of compound on the explant. Also, follow all precautions and take extensive care while working with the chemical.

• Hydrogen Peroxide: It’s also a dangerous chemical and rarely used for the sterilization process. Therefore, all precautions must be taken to sterilize the explant. The concentration most commonly used in the process is around 30%.
• Calcium hypochlorite: Dissolve the chemical in water and filter it before using it for the sterilization process. The concentration of the explant commonly used to clean the explant is 3.25%. The chemical has been found very effective for sterilization of Ziziphus spina-christi (L.) among other techniques.  
• Ethanol: Ethyl alcohol or ethanol is one of the extensively used and powerful chemicals for explant sterilization. However, only a few seconds of exposure must be given to the explant because it’s highly phytotoxic. Longer exposure time will lead to tissue damage. Typically, it is employed at a concentration of 70% (v/v) for a brief period of a few seconds or minutes, followed by a treatment with other chemicals. 

General Procedure to Sterilize Explants

The procedure of explant sterilization differs based on the type of explants being used for the tissue culture process. The duration of chemical exposure to the explant and the chemical used is also decided based on the nature of the explant. Because too much exposure to harsh chemicals to soft explants can damage their tissues.

Here’s a general explant sterilizing method to obtain infection-free explants for your tissue culture processes:

• Using tap water remove dust or any other external material attached to the plant part or tissue.
• Wash it again using distilled water.
• Now take some amount of distilled water in a beaker and add a few drops of detergent, such as Tween 20. Put the explant in the solution with continuously stirring.
• Rinse the explant twice using sterilized distilled water.
• Use a sharp scalpel to trim the size of the explant under the laminar hood.
• Surface disinfect the explants using 70% ethanol for 30 seconds. (Some procedures use HgCl2 (mercury chloride), NaOCl (sodium hypochlorite), and bromine water to disinfect explants).

Below is a table for different disinfectants used to sterilize explants, their preferred concentration, and the frequency with which they are used in the tissue culture process.

• Rinse the explant twice with sterile distilled water.
• Now, transfer the explant to a beaker containing 20% chlorox and sterilize it for 20 minutes. Continuously stir the explant for proper sterilization.
• Rinse the explant with three changes of sterile distilled water.
• Remove the explant and transfer it to a sterile culture medium.

Actions to Enhance Sterilization Procedures

Here're some steps following which can help you clean your explants better when combined with any explant sterilization methods:

• Adding a wetting agent, like TWEEN 20 or 80, to disinfectants can help reduce surface tension and improve contact with surfaces.
  
• One way to sterilize materials is to soak them in a solution made of 70% ethyl alcohol for a short time. After that, another disinfectant solution can be used to complete the sterilization process. For some species, it has been found that using a two-step sterilization procedure involving two different sources of sterilization can be advantageous.
• Performing the sterilization procedure under vacuum can effectively eliminate air bubbles and enhance the efficiency of the process.
  

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Happy Culturing!!