Browning in tissue culture media

Did you know that the browning of tissues and culture media is one of the major problems in plant tissue culture?

Although plant tissue culture labs try their best to maintain a sterile environment, in vitro contamination of cultures by microorganisms is often the most serious problem. Apart from contamination, there is another condition called 'browning' that poses a threat to the growth and development of plantlets. Browning of explants/excised plant tissues and culture media occurs frequently and remains a major reason for in vitro recalcitrance.

After reading this, if you are wondering what are the microbial contaminants that can ruin a tissue culture experiment, then do have a look at our article on "Different types of contaminants in plant tissue culture"

What is in vitro recalcitrance?

In vitro recalcitrance defines as the inability of plant cells, tissues, and organs to react to in vitro climate (e.g. nutrient media, temperature, humidity, etc.). This can pose a major limiting factor when you are trying to regenerate economically important plant species. It can also slow down or impair your efforts to apply in vitro conservation techniques in some species.

What is browning in tissue culture media?

When you make cuts in different plant parts in order to obtain desired explants, it causes wounding of tissues. This wounding further facilitates the release of various enzymes such as polyphenol oxidase, superoxide dismutase, and peroxidase in the wounded plant part. These enzymes come to the rescue of the plant by catalyzing various reactions to eliminate reactive oxygen and heal the plant part. One of these reactions results in the production of 'melanin', a dark pigment, which results in the browning of media and also explants.

When there is an accumulation of large quantities of melanin due to oxidation of phenolic compounds, it results in reduced plant growth, lower rates of regeneration or recalcitrance, and can finally lead to tissue/plant death.

Different phenolic compounds generally found in plants are:

  • Dopamine;
  • Catechin;
  • Chlorogenic acid;
  • Cinnamic acid;
  • Hydroxybenzoic;
  • Pyrogallic acid;
  • Salicylic acid;
  • Ferulic acid;
  • Vanillin;
  • Coumarin; and
  • Phenols.

Functions of different phenolic compounds in plants

The phenolic compounds produced in plants can be beneficial in different ways such as:

  • Maintaining plant healh by working as antioxidants.
  • Providing structural strength to the plant tissues.
  • Facilitating pollination by attracting pollinators and beneficial insects.
  • Providing defense against harmful radiations.
  • These compounds are produced as a result of plant defense mechanisms. Therefore, they also act as repellent to pests.
  • Improving plant's immune system against pathogens.

Even though phenolic compounds serve great benefits for the good growth and survival of plants, under in vitro conditions they tend to be detrimental. Browning caused by the oxidation of phenolics hinders tissue growth by reducing cell division rate and as a result, the plant does not grow further leading to failure of the experiment and plant death.

In vitro recalcitrance or tissue death is a major issue in the case of woody plant species among all other plant categories. This is because woody plants parts are more susceptible to secretion of higher levels of phenolic compounds from the cut or wounded ends of the explants.

Factors determining severity of browning

Different factors that determine the variation in browning severity under in vitro conditions are:

  • Plant species or variety/genotype;
  • Selection of tissues to be treated as explant;
  • Choice of plant organ as explant;
  • Developmental phase of the plant;
  • Age of tissue or organ;
  • Composition of nutrient medium; and
  • Other tissue culture variables like culture conditions, culture time, etc.

Browning can be influenced by environmental factors as well. According to scientific reports, the presence of light and high temperature raise the browning rate by increasing the enzyme activity.

Prevention of browning of medium

Several studies have been conducted by plant scientists to minimize the effect of phenolic compounds in tissue culture processes. As a result, many solutions have been formulated so far to deal with the browning contamination. Here is the list of commonly used treatments in a tissue culture facility:

Activated charcoal: Activated charcoal works by trapping toxins and inhibitory compounds and thereby preventing the absorption of these compounds by plants. You can add activated charcoal in the culture media and hence, reduce the accumulation of oxidized phenolic compounds. It also improves tissue growth and development.

Polyphenol inhibitors: Certain compounds or chemicals have the properties to inhibit the production of phenolic compounds in tissue culture conditions. Adding these inhibitors can show significant reductions in visual tissue browning, reduction in total phenol content which leads to a significant increase in tissue growth and development. Some examples are sodium chloride, sulfur dioxide, 2-aminoindane-2-phosphonic acid, etc.

Antioxidants: Adding antioxidants can reduce the amount of oxidation of phenolic compounds. This leads to a reduction in the browning of media. Common antioxidants are ascorbic acid, citric acid, polyvidone, cysteine, silver nitrate, etc.

Subculturing: You can perform frequent subculturing of explants/shoots to avoid browning. When subculturing is done frequently, it not only provides fresh nutrients and an environment for growth, but also avoids the problem of phenolic compounds. Thus, leading to improved growth and development of cultures.

Dark culturing: In principle, light promotes the production of phenolic compounds. Thus, culturing the explants in dark for about 72 hours to a period of 10 days can significantly reduce the chances of browning. However, the duration of culturing in the dark depends on the species under consideration.

These are some of the treatments which are popularly followed for dealing with the problem of browning. However, one treatment alone cannot ensure success. Therefore, it is wise to treat your cultures with a combination of these treatments to achieve a higher rate of success.

We hope you got a glimpse of different aspects related to oxidative browning. For more informational posts on different aspects of plant tissue culture, keep checking this space!

Also if you like this article, do share it with others as well. Let us build a wonderful community of plant tissue culture enthusiasts.

By Nancy Bhatia | 05-October-2021