13 Aug 2024

How to Sterilize Your Explants

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, simplify 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

Ask all tissue culturists, and they will tell you that maintaining an aseptic environment in your lab is the #1 requirement for tissue culture. 

If you want a higher success rate, you need to ensure everything is clean and sterilized, and yes that includes your plant tissues (also known as explants).

For cleaning your utensils, bench, and other equipment and tools, you’ve been advised to use disinfectants, ethanol, or dry and wet heat sterilization techniques like autoclaving and microwaving. But what about your explants?

Do you know what chemicals you can use to sterilize your explants? What processes do you need to follow?

Do you know how to determine which sterilization technique is most suitable for your explant? If not, then keep reading because these are the questions we are going to answer in this article.

What Is Surface Sterilization?

Surface sterilization of the explant is a crucial step in plant tissue culture, essential for preventing microbial contamination of the explant (the plant material used for culturing). It involves treating the plant material with an appropriate concentration of disinfectants, or sterilants, for a fixed duration to eliminate surface microorganisms without harming the internal tissues. 

By ensuring a sterile environment, surface sterilization greatly increases the success rate of plant tissue culture experiments. 

The sterilization process typically involves:

1. Initial cleaning: Remove visible dirt and debris from the explant using sterile water.
2. Detergent wash: Use a mild detergent to loosen contaminants.  
3. Disinfection: Immerse the explant in a disinfectant solution for a specific time.  
4. Rinsing: Thoroughly wash the explant with sterile water to remove disinfectant residues.

For comprehensive, step-by-step guidance on explant sterilization techniques, visitvisit this article to learn how to effectively sterilize your plant tissues for successful tissue culture.

Methods of Explant Sterilization?

Some chemicals used to sterilize explants include:

• Sodium hypochlorite: Commonly known as bleach, sodium hypochlorite is highly effective in killing bacteria at micromolar levels. Typically diluted to 10-20% for use in tissue culture, it requires 10-20 minutes for sterilization but is aggressive, making calcium hypochlorite a preferable alternative in some cases.
• Hydrogen peroxide: Although effective, hydrogen peroxide is rarely used in tissue culture due to its dangerous nature which can easily damage delicate plant tissue if not carefully controlled. It is typically employed at a concentration of around 30%, and extreme care must be taken to avoid tissue damage during the sterilization process.
• Calcium hypochlorite: Dissolved in water and filtered before use, calcium hypochlorite is used at a concentration of about 3.25%. It is favored for its effectiveness and is less aggressive than sodium hypochlorite.

• Ethanol (or isopropyl alcohol): Ethanol is a highly effective sterilizing agent, but it can also be toxic to plants. Typically, it is used at a concentration of 70% (v/v) for a brief period—just a few seconds or minutes—before being followed by other disinfectants.
• Benzalkonium chloride: This chemical is a quaternary ammonium compound that serves as a disinfectant and antiseptic. It is less commonly used for explant sterilization, but it can be effective at lower concentrations with a broad spectrum of antimicrobial activity.
• Silver nitrate: Silver nitrate is used for its antimicrobial properties, particularly against bacteria and fungi. It is less common in plant tissue culture, but can be used in specific protocols where bacterial contamination is a concern.
• Mercuric chloride: Highly toxic, mercuric chloride is rarely used due to safety concerns. When used, it requires multiple rinses with sterile distilled water to remove all traces, as even minute residues can be hazardous.
• Bromine water: Bromine water is an oxidative sterilant with potent antimicrobial effects. It is seldom used in plant tissue culture due to its reactive nature and potential to cause tissue damage.

Must Know: 

Disinfectants like ethanol and sodium hypochlorite (NaOCl) slow down the proliferation of fungi and bacteria in culture media. 

Hypochlorite, known for its potent antibacterial properties, can drastically reduce bacterial numbers even at micromolar levels. When dissolved in water, hypochlorite salts (NaOCl, CaOCl2) generate HOCl, with the effectiveness of bacterial elimination closely linked to the concentration of HOCl.

Factors Influencing The Effectiveness Of Surface Sterilization

Different plants and their parts require varying types, concentrations, and exposure times for disinfectants. Numerous surface sterilization techniques have been developed to clean explants, but the specifics of each method must be tailored to the particular plant species and tissues involved.

Here’s what influences the effectiveness of the sterilization technique:

• Type of explant: Different plant tissues vary in their susceptibility to contamination.
• Contamination level: The initial level of microbial contamination on the explant.
• Disinfectant concentration and exposure time: The strength and duration of the disinfectant treatment.
• Aseptic technique: Maintaining a sterile environment during the entire process.

By carefully following these steps and considering the specific requirements of the plant material, researchers can significantly reduce the risk of contamination and improve the success rate of their tissue culture experiments.

A Few Examples of Surface Sterilization of Explants

1. Surface sterilization of peach (Prunus persica)shoots

• Conduct sterilization procedures under aseptic conditions in a laminar airflow cabinet using sterilized instruments.
• Cut shoots into 1.5–2 cm pieces, ensuring each contains an axillary bud, and isolate the shoot tip (meristems with two or three primordial leaves).
• Thoroughly wash the explants (shoot tip and axillary buds) with tap water 3–5 times.
• Agitate the explants in liquid soap for 30 minutes to physically remove most microorganisms.
• Treat the explants with 70% ethanol for 1 minute, then rinse them three times with distilled water to minimize ethanol’s toxic effects.
• Treat the explants with varying concentrations of sodium hypochlorite (NaOCl) solution (10%, 20%, and 30% v/v) with 5.25% active chlorine for 15 minutes to determine the optimal concentration for each explant type and genotype.
• Add a few drops of "Tween 80" as a wetting agent to the NaOCl solution.
• Decant the sterilizing solutions under safe conditions.
• Wash the explants three times with autoclaved distilled water, 5 minutes per wash, to remove any traces of NaOCl.
• Surface sterilize the shoot tip and axillary bud explants.
• Trim the sterilized explants to a length of about 1–1.5 cm to expose a fresh-cut surface and eliminate any cells damaged during the sterilization process.

2. Surface Sterilization of Philodendron Species

The study followed the following protocol:

• Plant Material: Three self-heading cultivars of Philodendron (Imperial Green’, ‘Imperial Red’ and ‘Imperial Rainbow’) were selected for the study.
• Pre-treatment: The plants were treated with fungicides (Ridomil MZ and Mancozeb) for a week before the explants were taken.
• Preparation: After removing the leaves, shoot cuttings were washed and cut into single nodal segments, each containing a lateral bud.
• Surface Sterilization: The nodal segments were sterilized with ethanol (70%) and sodium hypochlorite (1% with Tween-20) to eliminate contaminants.
• Culture Medium: The sterilized explants were cultured on Murashige and Skoog (MS) medium, supplemented with specific plant growth regulators (NAA and BA), sucrose, and agar. The medium’s pH was adjusted before autoclaving.

3. Surface Sterilization of the seeds of Pokeweed (Phytolacca americana L.)

Harvesting and Preparing Seeds:

• Harvest pokeweed berries from mature plants.
• Separate the seeds from the pokeberries.

Breaking Seed Dormancy:

• Soak the seeds in concentrated sulfuric acid for 15 minutes.
• Rinse the seeds thoroughly with running tap water to remove the acid.

Surface Sterilization:

• Shake the seeds in a 12% (v/v) Clorox solution (containing 0.72% v/v sodium hypochlorite) for 45 minutes.
• Rinse the sterilized seeds with sterilized distilled water for five minutes, repeating the rinse at least three times.

Germination:

• Germinate the sterilized seeds on Murashige and Skoog (MS) medium, supplemented with 30 g/L sucrose.
• Adjust the pH of the medium to 5.8 before solidification with 8 g/L agar.
• Sterilize the medium by autoclaving at 121°C for 20 minutes.

Culture Maintenance:

• Maintain the cultures at 25 ± 2°C for six weeks under a 16/8 hour light/dark cycle with a light intensity of 40 μmol·m−2·s−1.

I hope these protocols give you an idea of how surface sterilization techniques differ based on the plant species and explant used. Therefore, to obtain a higher success rate in your tissue culture process, it's crucial to ensure that you develop an effective surface sterilization technique for your explant. 

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