13 Jul 2021

6 Most Common Problems in Tissue 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

Tissue culture is an advanced technique to produce plants rapidly in a mass, irrespective of their growing season. It’s a technical tool that requires some level of practical knowledge and skill. And, there are some problems that you will encounter more often if the experiment is not properly done, maintaining complete aseptic conditions.

Being aware and knowledgeable about problems beforehand makes you prepared to tackle them when you face them in real life. The most common challenges that you might face in your tissue culture lab include contamination, vitrification, browning of media, recalcitrance, somaclonal variation, and loss of totipotency.

So, in this article, we will cover the reasons behind these problems and their possible remedies. These can be very useful for the beginner culturists who are new to this field. Let’s start!

1. Contamination

Tissue culture requires a completely sterilized or aseptic environment. But, any negligence in maintaining the proper environment may lead to contamination of cultures. It will affect the time, money, and effort that you’ve put into culturing plants.

The most common microorganisms affecting cultures include fungi, bacteria, molds, and yeasts. In some cases, they are introduced from an outside source but in others, they might be residing inside associated with explant tissues, as endophytes.

How can you avoid it?

The culture room should be clean and the culture space should be organized, equipped with laminar flow, and sterilized with alcohol before working.
The consumables, culture vessels, media, and water should be sterilized.
The explants should be properly surface sterilized.
Use PPM (plant preservative mixture) like products to prevent contamination from the cultured plants.

2. Vitrification

It’s a physiological disturbance or malformation occurring in tissue culture plants and affecting their proper growth and development. In this case, the plants have glassy appearances and thick, enlarged, and brittle leaves and stems.

The main causes of vitrification in tissue culture plants are believed to be the factors triggering oxidative stress. It includes:

High salt concentration.
High relative humidity.
Low light intensity.
Gas accumulation in the atmosphere of the jar.
Length of time intervals between subcultures.
The number of subcultures, concentration, and type of gelling agent.
The type of explants used.
The concentrations of microelements.
Hormonal imbalances (high levels of cytokinins)

What’s the control?

Increase carbohydrate levels in the medium
Change light intensity.
Modify concentration of agar.
Reduce humidity in the culture container.
In some cases, an increase in calcium concentration has been observed to reduce the problem.

3. Browning of Media and explant

Tissue browning is the result of the accumulation and oxidation of phenolic compounds in culture tissues. It reduces the rate of cell division and regeneration capacity of explants that cause either their death or null responses in culture media. The phenomenon can be majorly observed in woody plant species.

How to prevent it?

Use activated charcoal in the media to avoid the accumulation of inhibitory phenolic compounds.
Use antioxidants and citric acid antioxidants in the media to prevent the oxidation of phenolic compounds.
Subculture the explants after a period of time to avoid the accumulation of the browning compounds.
Use a sharp-edge knife to cut the explants and keep them in water for a while, which act as the best solvent for secondary compounds present in tissues.
In some cases, explants placed in vertical positions also help against browning conditions.

4. Recalcitrance

Recalcitrance is defined as the inability of plant cells and tissues to respond in an in vitro culture environment. Tissue recalcitrance is generated because of the reaction of free radicals with the macromolecules of cells, causing cellular dysfunction.

How to avoid it?

Select juvenile parts of the plants as explant.
Rejuvenate parts of desired plants using cytokinin spray for tissue culture.

5. Somaclonal Variation

It is the phenotypic or genotypic changes in the cultured plant. It occurs due to change in chromosome structure and number; and DNA sequence. The phenomenon is termed genetic heterogeneity.

How to avoid it?

Avoid long-term cultures.
Prevent use of 2, 4-D in culture media.
Use axillary shoot induction systems.
Propagate chimeras by other clonal systems.
Keep the number of subculturing plants at a minimum.
Reinitiate cultures from new explants.

6. Acclimatization

Acclimatization is the adaptability of tissue cultured plants when brought to the natural environment or in the greenhouse. You must know, plants cultured in a completely artificial environment go under shock and die if not acclimated properly.

How to prevent it?

Optimize the hardening conditions for plants such as mineral nutrition in media.
Establish a photoautotrophic system.
Gradually adapt the plant in ex vitro conditions. Like, gradually adjusting the percentage humidity for in vitro plants when transferred to ex vitro conditions.

These are the 6 most common issues that you might face frequently in your tissue culture processes. Let us know if the content was useful to you and what more kind of tissue culture content you might want us to write about. We’ll be delighted to hear from you!

Happy Culturing!