13 May 2021

Prevent Your Explants from Browning Now!

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 Issue

The contamination is not the only problem in tissue culture; there are some other issues related to the tissue culture processes that you should be aware of. Remember, you can only find a solution when you understand the problem. Common culturing problems include fungal contamination, excessive hydrosis (vitrification), setting up the right composition of hormones, sugar, and other elements required by the plants, choosing the right explant for your cultures, browning of the medium, number of explants to be cultured on the media, etc.

This article talks about the browning of the media and its prevention, which will eventually lead to enhancing the productivity of your cultured plants.

Related: Tissue Culture Contamination

The woody plants and some herbaceous plants contain phenolic compounds in their cells and tissues. Some examples of phenolic compounds present in plants are dopamine, catechin, chlorogenic acid, cinnamic acid, hydroxybenzoic, pyrogallic acid, salicylic acid, ferulic acid, vanillin, coumarin, and phenols. The phenolic compounds benefit plants in several ways.

1. They can act as antioxidants
2. They provide (like lignin) structural strength to the plant tissues.
3. They act as attractants to insects and bees, who aid in pollination.
4. They help plants to fight against pathogens and pests.
5. They are involved in the defense against ultraviolet radiation.
6. Some phenolic metabolites provide coloring for camouflage.

Negative Effects of Phenolics

Apart from the wonderful benefits the phenolics compounds provide to plants, there is one negative side of it when it comes to tissue culture. The oxidation of phenolics in tissue culture harms the growth of tissues in the media and leads to browning of tissues and the growth medium. It reduces the rate of cell division and capacity of explant regeneration that eventually leads to the death of the cultured tissues and failure in the plant tissue culture.

Figure: The image showing (a) initiation of browning if explant, (b) complete browning of the explant and media.

Source: Babaei, Nahid & Abdullah, Nur Ashikin & Saleh, Ghizan & Abdullah, Thohirah. (2013). Control of contamination and explant browning in Curculigo latifolia in vitro cultures. 10.5897/JMPR012.859.

The problem is huge in the case of tissue culture of woody plant species, because of higher chances of exudation or leaching out of phenolic compounds from the cut or wounded ends of the explants. The rate of phenolic exudation depends on the season and presence of plant secondary metabolites. Several factors affect the browning of the medium including plant species and genotypes, explants and physiological status, explant damage, medium composition, and culture conditions, and culture time.

Prevention of browning of the medium

1. Use of activated charcoal in the growth media: Activated charcoals can efficiently absorb the inhibitory compounds from the media and reduce the accumulation of toxic metabolites, phenolic exudation, and brown exudate.
2. Use of polyphenol inhibitors in the media: Compounds that can inhibit the production and leaching of polyphenols can be very useful in the case of browning of the explants. It includes sulfur dioxide, sulfite, and sodium chloride.
3. Use of antioxidants in the growth media: Most frequently used antioxidants in labs to prevent the browning of the media are ascorbic and citric acid. The antioxidant reduces the oxidized substrate to prevent the browning of the media or explant. Prepare fresh solutions each time before initiating the culturing process. In Musa paradisiaca, potassium citrate has been found to be a better antioxidant against the darkening of the media. Some other antioxidants include ascorbic acid, cysteine, butylated hydroxyanisole or butylated hydroxytoluene, and PVP.
4. Frequent subculturing of explants: The fast subculturing of the explants can avoid accumulation and penetration of the phenolic compounds in the media and explants. Thus, enhance the productivity of the cultures.
5. Prefer culturing in the dark that prevents polyphenolic exudation: Light stimulates the synthesis of polyphenolic compounds. So, placing the explants in the dark for about 72-96 hours (depending on the species) may help to reduce the browning. However, the technique might not work for all species of the plant. A combination of this technique with other effective techniques of preventing browning can be a good idea! But remember, different hours of treatment will be required for different plants.
6. After the excision, soak explants in the water: Use a sharp-edged knife to cut the explant, this helps to reduce the wound. Also, after explant excision, keep it in the water, it’s the best solvent for the secondary compounds present in the tissues. It can be an effective way to reduce browning.
7. The position of explants in the culture media: It has been observed in several studies that after excision when explants are placed vertically, rather than horizontally, on the culture media, they give better results against browning condition.

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Additional Methods

Apart from the above-mentioned technique to prevent browning of the media and explant, some other methods include:

1. Supplement the media with compounds like adenine sulfate and 300 mg L–1 polyvinylpyrrolidone (PVP) that controls the polyphenolic exudation.
2. Seal the cut ends of explants to prevent the leaching of polyphenolic compounds.
3. Use a growth media having lower salt concentration.

To enhance productivity and prevent the browning of the media to the maximum extent, it’s better to use the combination of one or more of the above-mentioned techniques. Also, note that the efficacy of the technique differs depending on the species of plant and explants you use. The concentration of the above-mentioned compounds that help prevent the browning of the media and explant also depends on the same factor.

Let us know if you found the techniques helpful or if you are trying some other ways to reduce the browning of your media and explant. We’ll be excited to hear from you!

Happy culturing!

References

1. Bhatia, S., & Sharma, K. (2015). Technical Glitches in Micropropagation. Modern Applications of Plant Biotechnology in Pharmaceutical Sciences, 393–404. doi:10.1016/b978-0-12-802221-4.00013-3
2. Babaei, Nahid & Abdullah, Nur Ashikin & Saleh, Ghizan & Abdullah, Thohirah. (2013). Control of contamination and explant browning in Curculigo latifolia in vitro cultures. Journal of Medicinal Plants. 7. 448-454. 10.5897/JMPR012.859.
3. https://pubmed.ncbi.nlm.nih.gov/18786626/
4. https://e-repository.org/rbl/vol.16/iss.6/10.pdf
5. https://scialert.net/fulltext/?doi=ajbs.2018.165.1...
6. https://iopscience.iop.org/article/10.1088/1757-89...