30 Jul 2024

Common Tissue Culture Problems And How To Deal With Them (Part 1)

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

Tissue culture is undoubtedly an excellent technique for growing plants.

You take a piece of the plant, provide it all essential nutrients, and keep it in a condition that supports its growth and development. 

It sounds easy, right?

But is the technique really that straightforward when put to application?

The answer depends on the level of your expertise in working with the plant. If you're an expert and have found a perfect recipe and physiological parameters that support plant growth, your success rate will likely be higher. 

Your familiarity with potential problems and their solutions plays a significant role.

However, if you’re a beginner—perhaps experimenting with tissue culture as a hobby at home or integrating it into your plant business for the first time—it might not be so easy.

You could encounter several challenges in growing your plants.

“What kind of challenges?”, you ask.

Read on in this article to discover the answers.

The Potential and Challenges of Plant Tissue Culture

Plant tissue culture (PTC) is a revolutionary technique that has transformed the field of horticulture and agriculture. It involves growing plants from small tissue samples in a controlled environment, providing them with essential nutrients and optimal growth conditions. This method offers several promising techniques, such as in vitro micropropagation, organogenesis, and somatic embryogenesis.

Applications of Plant Tissue Culture

PTC has a wide range of applications that make it invaluable to modern agriculture and horticulture:

1. Pathogen-Free Plant Production: PTC can produce plants free from pathogens, ensuring healthier crops and higher yields.
2. Somatic Hybridization: This technique allows the combination of different plant species, creating hybrids with desired traits.
3. Rapid Propagation: It enables the quick propagation of plants that are difficult to propagate through traditional methods.
4. Genetic Improvement: PTC aids in the genetic enhancement of commercial plants, allowing the incorporation of desirable traits.
5. Haploid Plant Production: It facilitates the production of androgenic and gynogenic haploid plants, shortening breeding programs.
6. Conservation of Rare Plants: PTC plays a crucial role in conserving rare and endangered plant species.
7. Abiotic Stress Tolerance: It helps in developing plant varieties that are tolerant to abiotic stresses like drought, salinity, and heat.
8. Production of Bioactive Compounds: PTC can be used to produce secondary metabolites and bioactive compounds, especially through plant-cell-suspension cultures.

The economic implications of PTC are substantial. Horticultural nurseries, in particular, rely heavily on this technology to produce high-quality planting materials.

8 Most Common Plant Micropropagation Challenges

Despite its advantages, PTC is not without its challenges. Several obstacles can cause significant losses in in vitro cultured plants: contamination, hyperhydricity, browning and phenol exudation, shoot tip necrosis, somaclonal variations, root hardening, and failure of acclimatization.

Let’s talk about these obstacles in detail and learn how to deal with them.

1. Contamination

The primary requirement of tissue culture is maintaining an aseptic environment. Since the nutrient media contain sugar, the chances of microbes invading the cultures are higher. Therefore, it’s required to clean your hands and sterilize the culture area and all the equipment and chemicals you use in the process. 

Microbes are ubiquitous, present in air, water, and on surfaces. They can enter your lovely cultures from anywhere. Therefore, a strict aseptic environment is necessary for the success of plants in tissue culture. 

If ignored, contamination can lead to the loss of your cultures, waste of your efforts, and valuable resources invested in the process. 

So, how can you avoid it?

You can do this by following general best practices to maintain an aseptic environment and use of PPM™ (Plant Preservative Mixture)™ in your media. 

2. Browning

Phenolic compounds, naturally occurring in many plants, can pose a significant challenge in tissue culture. These compounds, when oxidized, lead to browning of the explant and culture medium, inhibiting plant growth and development. This phenomenon is particularly pronounced in woody plants due to their higher phenolic content.  

Several factors contribute to browning, including plant species, explant type, culture conditions, and the presence of light. To mitigate this issue, various strategies can be employed:  

• Utilize activated charcoal: This adsorbent effectively removes phenolic compounds from the culture medium.  
• Incorporate antioxidants: Ascorbic acid and citric acid are commonly used to inhibit the oxidation process.  
• Employ polyphenol inhibitors: Compounds like sulfur dioxide and sodium chloride can reduce phenolic exudation.
• Practice frequent subculturing: Regular transfer of explants can prevent phenolic compound accumulation.
• Optimize light conditions: Reducing light exposure can minimize phenolic synthesis.
• Pre-treat explants: Soaking explants in water can leach out some phenolic compounds before culturing.
• Consider explant orientation: Vertical placement of explants may reduce browning in some cases.  

By carefully considering these approaches and tailoring them to specific plant species and culture conditions, researchers can effectively minimize browning and enhance tissue culture success.

3. Hyperhydricity

Hyperhydricity (HH) is a physiological disorder affecting in vitro plants, characterized by excessive water content, reduced lignification, and a translucent, often fragile appearance. 

This condition significantly impacts plant growth, development, and ultimately, the success of tissue culture protocols. HH can lead to decreased multiplication rates, reduced plant quality, and hindered acclimatization.

Several factors contribute to HH, including high humidity, imbalanced nutrient levels (particularly calcium and ammonium), inadequate gas exchange, and the use of certain gelling agents like gelrite. To mitigate HH, consider the following strategies:

• Optimize culture conditions: Ensure adequate ventilation, light intensity, and humidity levels.
• Adjust medium composition: Balance nutrient concentrations, especially calcium and ammonium. Explore alternative gelling agents like agar, Danish agar, or Cero Agar.
• Utilize growth regulators: Incorporate growth regulators like salicylic acid, spermidine, or hydrogen peroxide to regulate plant physiology.
• Control environmental factors: Minimize ethylene accumulation by frequent subculturing and proper ventilation.

4. Somaclonal Variation

Somaclonal variation, the genetic instability observed in tissue culture-derived plants, poses a significant challenge to the propagation of clonal genotypes. Prolonged exposure to in vitro conditions, often necessitated by repeated subculturing, is a primary factor contributing to this phenomenon. 

Studies have shown a direct correlation between increased subculture frequency and the incidence of somaclonal variation in species such as Tetrastigma hemsleyanum and Moringa oleifera.

Several strategies can be implemented to mitigate somaclonal variation. The addition of growth regulators like salicylic acid to culture media has shown promise in reducing its occurrence. 

Furthermore, proper handling of plantlets during transfer is essential to prevent physical damage, such as burns from hot instruments, which can exacerbate genetic instability.

In the next section of this article, we will discuss four additional challenges that impact the success rate of plants in tissue culture and how to address them. You will also discover five tips to enhance tissue culture productivity.

How is Plant Cell Technology Helping Culturists Worldwide In Their Tissue Culture Application?

Plant Cell Technology is helping tissue culturists around the world by providing unique and world-class products and services that smoothen their process. It has MS media, agar, gellan gum, Plant Preservative Mixture™ (PPM)™, culture vessels, Biocoupler™, and portable laminar hood in its store to facilitate your processes.

And, there’s more! 

Plant Cell Technology also offers consultation services to culturists of all sizes that help to get instant solutions to your tissue culture problems. Further, you can also join our live or on-site Master Class to directly learn from our expert and be pro in performing the tissue culture technique. 

So, visit www.plantcelltechnology.com today and find out more about our products and services and how we help you to excel in your tissue culture processes.

Happy Culturing!!