20 Nov 2024

Which Parts of the Plant Are Best for Tissue Culture and How Should They Be Sterilized?

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

What factors determine the success of plant tissue culture? Ultimately, while multiple variables come into play, a few critical ones stand out:

• A well-optimized working protocol
• A nutrient-rich media tailored to the plant species
• A healthy and disease-free mother plant
• A sterile, contamination-free work environment
• Proper physiological conditions
• Gradual and suitable acclimation of plantlets

At the initiation phase, however, the most vital factor driving success is the “explant” you choose.

An explant refers to any part of a plant—such as its leaves, roots, stems, or buds—selected from the mother plant. The tissue culture process cleans, sterilizes, and uses this tissue to regenerate a new plant under controlled conditions.

The choice of explant is pivotal because different plants respond uniquely to different parts used for culture.

For instance, in some species, leaf explants might yield better regeneration rates, while others might require meristematic tissues like buds or root tips. The physiological state of the explant also plays a role—factors like age, health, and active growth stages influence its ability to regenerate.

Sterilization of the explant is critical, as contamination can easily derail the entire process. 

Proper sterilization involves removing debris and microbes through surface sterilants like alcohol, bleach, or hydrogen peroxide, followed by careful rinsing to avoid chemical residues.

By meticulously selecting and preparing the explant, you set a strong foundation for successful tissue culture, ensuring healthy plant regeneration and high yield in subsequent stages of the process.

This article expands on the types of explants used for inducing plant regeneration in vitro and the best ways to sterilize and prepare them for the tissue culture journey. 

Types of Explants Used in Tissue Culture

In tissue culture, an explant is any plant part used to regenerate a plant under sterile conditions. Choosing the right explant depends on the plant species, the type of culture being performed, and the goal of the process. 

Common types of explants and their applications include:

1. Axillary or apical meristems: Growers prefer these young, healthy tissues, such as shoot tips or nodal segments, for micropropagation due to their high success rate in establishing cultures.
2. Leaves: Leaf segments are widely used for callus formation, particularly in dicots like tobacco, tomato, and potato. The right conditions can induce new shoots from callus.
3. Immature embryos: Commonly used in monocots, immature embryos are ideal for micropropagation and genetic studies.
4. Roots: Rarely used due to microbial contamination risks and the challenge of surface sterilization, but viable under specialized conditions.
5. Seeds or ovules: Effective for plants with low seed viability, this method ensures clean explants by sterilizing whole seeds or fruits.
6. Anthers and pollen: These are used to produce haploid plants, beneficial for genetic research and crop improvement.
7. Protoplasts: Plant cells without cell walls, obtained enzymatically or mechanically, are used for cell cloning, hybridization, and genetic transformations.

Each explant type requires specific preparation and nutrient media to achieve successful plant regeneration, emphasizing the importance of selecting explants based on plant physiology and culture objectives.

Choosing The Right Explant for Your Tissue Culture Process

Here are some factors that you should consider while selecting a suitable explant for your tissue culture process:

1. Age of Explant
   Juvenile tissues such as zygotic embryos and shoot tips are highly responsive due to their vigorous growth phase. For instance, seed-derived shoot tips often yield better outcomes in species like Acacia and Prunus. Juvenile tissues are more adaptable and show robust regeneration compared to mature tissues.
2. Source and Type of Explant
• Seeds and Embryos: Seeds protect the zygotic embryo, a highly regenerable plant part, making them a popular choice for micropropagation in hardwood species.
• Shoot Meristems: Growers prefer these for virus-free plant production and achieving high success rates in micropropagation.
• Leaf and Node Segments: Commonly used for clonal propagation and callus formation, particularly in mature trees. For example, nodal segments of Quercus and Wrightia have shown effective shoot induction.
3. Season of Collection
   The timing of explant collection influences success. Spring is often ideal for explant harvesting due to optimal hormone and nutrient levels. However, some species, like Ziziphus spina-christi, perform better with explants collected during summer.
4. Plant Health and Genotype
   Explants from healthy, disease-free plants respond better to tissue culture. Additionally, plant genotypes significantly affect their in vitro performance. Some species or cultivars are naturally more amenable to propagation techniques.
5. Purpose of Culture
   The choice of explant should align with the culture objective:
• Micropropagation: Shoot tips or nodal segments.
• Callus Formation: Leaf, cotyledon, or stem segments.
• Haploid Production: Anther or pollen grains.
6. Explant Size and Location
   Larger explants with nutrient reserves tend to perform better than smaller ones. Additionally, the growth regulator levels can vary depending on the explant's position on the plant (e.g., base vs. tip of a stem).

Mature vs. Juvenile Explants

While juvenile explants are typically more effective, mature tissues, such as nodal segments, can also yield promising results under in vitro conditions. In cases like Wrightia tinctoria and Dalbergia latifolia, nodal cultures have enabled successful cloning of mature trees. Mature tissues often undergo rejuvenation in culture, mimicking juvenile-like responsiveness.

Special Applications

• Seasonal Influences: Tropical species like Azadirachta indica and Artocarpus heterophyllus thrive with spring-harvested explants, while some others favor summer conditions.
• Callus and Organogenesis: Even mature explants can dedifferentiate into callus and redifferentiate into shoots under proper culture conditions, extending their utility for tissue culture processes.

Surface Sterilization of Explants—Chemicals and Methods Used

Surface sterilization is essential in plant tissue culture to eliminate microorganisms that can compromise the success of the culture. These microbes, found on surfaces, in crevices, and even within plant tissues, must be effectively removed while preserving the explant’s viability. 

Below is a comprehensive breakdown of the process:

1. Initial Cleaning with Soapy Water

The explant should first be washed in warm, soapy water to remove visible dirt, dust, and soil. This step minimizes the microbial load and ensures improved contact between the explant and disinfectants applied later. 

Adding a surfactant, such as a mild detergent like dish soap or Tween-20, helps break down waxy coatings on plant surfaces, further exposing hidden contaminants.

2. Pre-Treatment with Disinfectants

To ensure a sterile surface, the explant is treated with various disinfecting agents:

• Sodium Hypochlorite: Found in household bleach, this is diluted to concentrations between 5–25%. The solution is agitated for 5–30 minutes to maximize contact. Sodium hypochlorite is highly effective but can degrade over time, so fresh preparations are recommended.
• Calcium Hypochlorite: This alternative to sodium hypochlorite is used at 0.8% concentration. The solution is prepared by allowing the calcium hypochlorite to settle, decanting the clear liquid, and combining it with water for application.

3. Use of Alcohol for Sterilization

Ethyl or isopropyl alcohol, diluted to 70%, can be used for swabbing or dipping plant material. It is particularly useful for quick sterilization before or after exposure to other disinfectants. Alcohol disrupts microbial cell membranes, providing an additional layer of sterilization.

4. Hydrogen Peroxide Application

Hydrogen peroxide (3–10%) acts as a potent oxidizer, effectively killing surface microbes. It can be used alone or alongside other agents, but thorough rinsing is crucial to prevent interactions that might damage plant tissues.

5. Physical Techniques for Enhanced Sterilization

For plants with complex surface structures like hairs or waxy coatings:

• Sonication: Using ultrasonic waves for 2–20 minutes loosens debris and dislodges microbes from hidden crevices.
• Vacuum Evacuation: Air bubbles trapped in fine hairs or grooves can be removed under vacuum, allowing disinfectants to reach otherwise inaccessible areas.

6. Thorough Rinsing with Sterile Water

Following disinfection, the explant must be rinsed 3–5 times with sterile water. This step ensures the removal of residual chemicals, which could harm the plant tissue or interact negatively with the culture medium.

7. Selection of Disinfectant and Timing

The choice of disinfectant and its exposure time should balance efficacy and safety. Overexposure to strong chemicals can damage explants, requiring the removal of injured tissue before culturing. Trial and error, research, or study references can help optimize these parameters.

8. Managing Contamination from the Explant Source

Plants grown in greenhouses or controlled environments are often cleaner than those grown outdoors. Tissues taken during active growth periods, such as spring shoots, typically harbor fewer contaminants than dormant tissues or those in contact with soil, like roots or bulbs.

9. Dealing with Internal Microbial Contaminants

Even with thorough surface sterilization, microbes within vascular systems or intercellular spaces can persist. Juvenile tissues or actively growing shoots are less likely to contain internal contaminants, making them ideal explant sources. However, the use of antibiotics or heat treatments can help manage internal microbial loads in problematic cases.

Surface sterilization is a meticulous process that requires attention to detail and adaptability. 

By understanding the characteristics of the explant and tailoring the sterilization approach, contamination risks can be significantly reduced, paving the way for successful tissue culture outcomes.

Want Disease-Free Plants? Meristem Culture is Key—Learn How PCT Can Help You Perfect This Technique.

Plant Cell Technology (PCT) is a vital partner for those aiming to master meristem culture and produce disease-free plants. We invite you to join our online tissue culture master class, where we cover everything you need.

This comprehensive course covers all aspects of tissue culture, from contamination control to specialized methods like meristem culture. These techniques are designed to help you produce disease-free plants with precision.

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