6 Aug 2024

Common Tissue Culture Issues And How To Deal With Them (Part 2)

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

In the previous part of this article, we explored the potential of plant tissue culture (PTC) and some of the common challenges that can hinder its success. We talked about how contamination, browning, hyperhydricity, and somaclonal variations can affect your cultures and how you can deal with them. 

Learning these challenges is crucial to understanding what solutions you can implement to achieve success in your tissue culture operations. 

Here we discuss four more problems, including recalcitrance, shoot distortion, root hardening problems, and acclimation stage issues, that you might face in your experiments and how you can deal with them.

5. Recalcitrance

Recalcitrance, which is the inability of plant tissues to respond to in vitro culture conditions, remains a significant hurdle in plant propagation. This phenomenon is often observed in mature plant tissues, which are less responsive to regeneration compared to juvenile ones. Factors contributing to recalcitrance include genetic predisposition, the physiological state, and the presence of inhibitory substances.

To overcome recalcitrance, researchers have explored several strategies:

• Explant selection: Utilizing juvenile tissues or specific plant organs with higher regeneration potential.
• Hormone optimization: Manipulating growth regulator levels to induce morphogenesis.
• Culture medium modification: Adjusting nutrient composition and osmotic conditions.
• Preculture treatments: Employing stress or wounding to stimulate regeneration.
• Tissue manipulation techniques: Using thin-cell layers or protocorm-like bodies to isolate totipotent cells.
• Genetic engineering: Modifying gene expression to overcome recalcitrant responses.

6. Shoot Distortion

Shoot distortion, a prevalent issue in plant tissue culture, manifests in various forms, including basal callus formation and leaf abscission. Basal callus, an overgrowth of undifferentiated tissue at the shoot base, often results from imbalanced hormone levels, particularly excessive auxin. Leaf abscission, characterized by premature leaf drop, is frequently associated with ethylene production and cell wall degradation. Both phenomena hinder efficient plant propagation and reduce plantlet quality.

Strategies to Mitigate Shoot Distortion

• Control hormone levels: Maintain an optimal balance of auxins and cytokinins.
• Reduce auxin accumulation: Apply growth regulators judiciously.
• Prevent callus formation: Use appropriate explant types and culture conditions.
• Manage ethylene levels: Use ethylene inhibitors or reduce ethylene production.
• Optimize environmental conditions: Provide adequate ventilation, humidity, and light.
• Strengthen plant tissues: Use antioxidants or growth regulators to enhance cell wall integrity.

7. Root Hardening

Successful micropropagation hinges on the ability to establish robust root systems in vitro. However, inducing and developing healthy roots often presents significant challenges. Factors such as hormone imbalances, nutrient deficiencies, and environmental conditions can impede root formation and development. Moreover, the transition from in vitro to ex-vitro environments can be stressful, leading to poor acclimatization and reduced plant survival.

Strategies for Enhancing Root Development and Hardening

• Optimize hormone balance: Carefully regulate auxin and cytokinin levels to stimulate root initiation and growth.
• Adjust nutrient composition: Modify macronutrient and micronutrient concentrations to support root development.
• Improve culture conditions: Ensure optimal light, temperature, and humidity for root growth.
• Gradual acclimatization: Introduce plants slowly to ex vitro conditions to reduce stress.
• Utilize growth-promoting microorganisms: Use beneficial bacteria or fungi to enhance root health.
• Employ supporting materials: Use appropriate substrates (e.g., agar, rockwool) during acclimatization.

8. Proper Acclimatization

Acclimation is the critical process of adapting tissue-cultured plants to the challenges of the external environment. Raised in sterile, controlled conditions, these plants possess limited resilience to the fluctuations in light, temperature, humidity, and pathogen pressure encountered in greenhouses or fields. Consequently, a gradual acclimatization period is essential for their survival and growth.

Best Practices for Acclimation:

• Gradual Exposure: Increase exposure to external conditions stepwise.
• Humidity Control: Maintain high humidity initially, gradually reducing it.
• Light Management: Provide shaded conditions initially, gradually increasing light intensity.
• Hardening Off: Expose plants to varying temperature and humidity conditions.
• Nutrient Management: Ensure adequate nutrient supply in the new environment.
• Disease Prevention: Implement measures to protect against pests and diseases.
• Regular Monitoring: Observe the plants closely for signs of stress or adaptation.
• Patience: Allow sufficient time for the plants to adjust to new conditions.

How Can You Ensure Greater Success In Your Tissue Culture Operations?

Here are some tips to ensure you get better results from your tissue culture efforts:

• Explant Selection: Carefully choose healthy, young explants from disease-free plants.

Here are some factors to consider when choosing an explant for your plant tissue culture procedures:

• Explant age: Younger tissues often exhibit better growth responses and withstand sterilization.
• Explant collection time: Seasonal variations affect explant responsiveness and contamination risk.
• Explant size and location: Larger explants with adequate nutrient reserves are generally preferred. Explant position within the plant can influence hormone levels.
• Plant health: Healthy donor plants yield better explants.
• Culture purpose: Different explants are suitable for various tissue culture objectives (cloning, callus induction, haploid production).
• Plant genotype: Genetic variations influence explant response to culture conditions.

• Chemical Quality: Use high-quality, reagent-grade chemicals. Optimize sterilization times and concentrations based on explant characteristics.
• Hormonal Balance: Experiment with different hormone combinations and concentrations to find the optimal formula for your plant species.
• Aseptic Techniques: Adhere to strict aseptic protocols, including regular lab cleaning, proper attire, and the use of sterilization techniques.
• Gradual Acclimatization: Slowly introduce tissue-cultured plants to external conditions, providing adequate humidity, light, and ventilation.

How Can Plant Cell Technology Help You Tackle Your Tissue Culture Challenges?

We understand the challenges of setting up a home lab and mastering plant tissue culture. Plant Cell Technology is committed to supporting your journey with essential supplies, expert knowledge, and a vibrant community forum.

Whether through detailed blog articles, comprehensive master classes, or personalized consultation, we aim to empower your success in the dynamic world of plant tissue culture.

How Plant Cell Technology Empowers You:

• Essential Supplies: Access a comprehensive range of high-quality media, agar, gelling agents, culture vessels, and more, ensuring you have the right tools for every step.
• Expert Knowledge: Gain confidence through our informative resources:
• Comprehensive blog articles: Explore topics from basic principles to advanced techniques.
• Master Classes: Immerse yourself in in-depth sessions led by experienced instructors, gaining valuable hands-on experience and mastering advanced techniques.
• Consultation Services: Get personalized advice and tailored solutions from our experts, ensuring your home lab journey flourishes.
• Community Connection: Join our vibrant community forum to connect with fellow enthusiasts, share experiences, and stay updated on the latest advancements.