Hyperhydricity in tissue culture and methods to deal with it

Propagating plants is a rewarding and enjoyable process. It does not matter if you are an expert or if you are just starting out, when it comes to plant tissue culture nothing is granted. Even with the best supplies and the healthiest explants, problems are unfortunately bound to arise sooner or later.

Let us today discuss about one of the most common nightmares in plant tissue culture ‘hyperhydricity’.

What is hyperhydricity?

The term “hyperhydricity” refers to an undesirable physiological and morphological disorder. It occurs in a wide range of woody and herbaceous plants when cultured in vitro.

Hyperhydric plants in tissue culture appear glassy, the stem tends to be thick, and leaves seem translucent and may sometimes look curly and brittle as well.

Hyperhydric plants develop shoots that are unable to regulate their water balance and as a result, accumulate more water than necessary. The exact mechanism of how this problem is related with the visible abnormalities of the plants is still unclear. Hyperhydric plants show biochemical characteristics such as reduction in dry weight and in certain biochemical compounds present in plant cells, such as lower amounts of lignin, cellulose, and chlorophyll.

Why is hyperhydricity a problem?

Hyperhydric shoots often do not seem abnormal and continue growing in tissue culture media. You can easily damage the affected shoots by desiccation, and they might not survive when you perform subculturing or transfer them to an external environment. In most of the severe cases, there is a bad impact on the shoot tips and therefore, the shoot development stops.

The use of liquid medium has many advantages under in vitro culture. It is an ideal technique for mass production as it reduces manual labor as well as facilitates changing the medium composition. However, the major demerit of a liquid medium is hyperhydricity.

Hyperhydricity is not always a death sentence for plantlets, but it is a problem for tissue culture labs globally. This condition can occur when you are creating a new working protocol or even when you are working with an already optimized protocol. Some of the most economically important crops such as potato, carnation, aloe, sunflower, and apple are highly susceptible to hyperhydration.

Causes of hyperhydration

Some causes for hyperhydricity in plants are:

• High relative humidity inside the vessels;
• Ethylene accumulation (Ethylene is a plant hormone which controls growth and senescence of plants);
• Type and concentration of the gelling agents;
• Type and concentration of cytokinin; and
• Concentration of other nutrients in the media.

Overcoming hyperhydricity is a crucial part for a successful micropropagation protocol. Since a wide range of plants are affected by hyperhydricity, several studies have been conducted to find suitable ways to deal with it.

How to deal with the problem of hyperhydricity?

The development and growth of plants in a lab are largely dependent on different conditions that influence the microenvironment inside culture vessels. You can read more about them here “4 conditions for microenvironment in plant tissue culture”.

Having a large number of hyperhydric plants in a lab can cause many problems, especially when you are working with a plant genotype that has not been widely studied before.

However, you can still try double-checking these “hacks” in order to save your plants:

Ventilation in the vessels: In plant tissue culture, you grow plantlets inside sealed vessels in water enriched medium. Using ventilated vessels such as our inVenti⁺ cultivation vessels with filter which allows a better gaseous exchange and helps in minimizing the hyperhydration of cultured shoots. By providing efficient gaseous exchange, plants can acclimate rapidly and show healthy growth. 

Light intensity: The optimal light intensity depends on the specific plant species that you want to tissue culture. A non-suitable light intensity can stress shoots and increase the probability of hyperhydration. You can avoid hyperhydricity by using optimal light intensity as well as providing good ventilation in the culture vessels. This combination works by improving gas exchange inside the vessels and preventing the production as well as accumulation of ethylene.

Temperature: The growth room should also have an adequate temperature for shoot proliferation. The propagation of different species may require changing the temperature settings. If you are propagating a tropical plant and the temperature setting is low (<24°C) plants tend to hyper hydrate. A similar situation may occur for plants that naturally grow in colder conditions.

Culture media

• Nutrient balance: Choosing the most suitable media is vitally important for explants growth. The formulation of the nutrient medium remains an important part of the development for all applications of plant tissue culture. The most common media used in plant tissue culture is Murashige and Skoog, however there are some others that could be useful such as: WPM, Gamborg, or DKW. Studies have shown that adjusting media components, such as decreasing concentration of nitrogen and increasing concentration of calcium, iron, magnesium, can be helpful to overcome hyperhydricity.

• Concentration and type of gelling agent: Increasing the concentration of the gelling agent or using a different one has been helpful to decrease hyperhydricity in cultures. There are many different kinds of gelling agents available in the market. This hack works mainly because adding more gel decreases the availability of water and limits the water uptake.

• Cytokinin: This specific plant hormone tends to induce hyperhydricity in many species such as banana, cacti, and cannabis. High concentrations of cytokinin in culture media along with non-suitable microenvironment conditions contribute to the production of hyperhydric shoots. In most cases, a balance of the cytokinin concentration is enough to slow down hyperhydration as well as to maintain shoot proliferation.

• Exogenous additives: Youcan also experiment with different additives such as salicylic acid, PEG, and polyamines. These can successfully reverse the hyperhydricity in specific cultures. Couple of studies have shown promising results and might be a key for the future control of hyperhydricity.

Hyperhydricity is a common problem, and it has several methods to deal with it. Some of the procedures mentioned here may work for certain plant species and it is difficult to apply them in others. Different groups of plants have special needs and hyperhydricity in specific plant species should be studied individually.

We hope this article gives you a better understanding of photoautotrophic micropropagation. For more informational posts on different aspects of plant tissue culture, keep checking this space!

By Nataly Sánchez Del Río | 28-December-2021

References

• Cassells, A. C. (2003). TISSUE CULTURE | micropropagation. In B. Thomas (Ed.), Encyclopedia of Applied Plant Sciences (pp. 1353–1360). Elservier. https://doi.org/10.1016/B0-12-227050-9/00214-3
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• Ivanova, M., Van Staden, J. (2011).  Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla . Plant Cell Tiss Organ Cult 104, 13–21. https://doi.org/10.1007/s11240-010-9794-5
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• Martinez, L. & Visser, Richard & De Klerk, Geert-Jan. (2010). The hyperhydricity syndrome: Waterlogging of plant tissues as a major cause. Propagation of Ornamental Plants - PROPAG ORNAM PLANTS, 10(4), 169-175.
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• Van den Dries, N., Gianni, S., Czerednik, A., Krens, F. A., & de Klerk, G.-J. M. (2013). Flooding of the apoplast is a key factor in the development of hyperhydricity. Journal of Experimental Botany, 64(16), 5221–5230. https://doi.org/10.1093/jxb/ert315