4 conditions for microenvironment in plant tissue culture
All plants require the right environment, be it in a field or a laboratory. But did you know about the conditions you need to maintain for the microenvironment in plant tissue culture?
Though the genetic makeup of a plant species has a large influence on the growth and development under tissue culture conditions, the success of in vitro propagated plant tissues is largely determined by environmental conditions.
Plants growing in the field also interact with, and adapt to the surrounding environment to thrive or survive. This is one of the major reasons for extensive plant breeding research. Multiple varieties are developed and optimized to grow in certain climatic conditions. For example, for a better yield, or in order to grow in a different climate the species is originated from.
Plants in a laboratory are fragile and therefore even more sensitive to the environment. However, the advantage of in vitro propagation is that the environment is easier to control once the optimal conditions are there. To find these optimum conditions we need to answer multiple questions. For instance, which light spectrum, light intensity, and day-night schedule would suit a certain plant best? Which plant growth hormone and in what quantities would lead to apt growth? And so on.
When you are reading about optimal conditions to grow plants in tissue culture facilities, then you might also want to know more about how to build a laboratory. You can read about different pieces of equipment you need for a fully functioning lab in our article on "How to set up a tissue culture laboratory?".
Let us now talk about different conditions for the microenvironment in plant tissue culture:
What does maintaining microenvironment in plant tissue culture means?
It means providing optimum conditions to the plant's cells/tissues for developing with the help of tissue culture techniques. It primarily involves physical environmental conditions such as light, temperature, humidity, etc. in the culture room as well as culture vessels. But it also involves a suitable nutrient composition in culture media for efficient growth and development of plantlets.
In places like greenhouses or even fields, plants have to compromise with the environmental conditions. Not all plant species can keep up with extreme climatic fluctuations. Some of the plant species grow well only in tropical conditions while some survive only in colder conditions. On the contrary, when we develop plants in closed culture vessels, we are providing them an optimal environment. Hence, these vessels serve as a barrier between the internal environment and the external environment.
So what are the conditions that influence the microenvironment? These are:
- Gaseous exchange/air movement;
- Temperature; and
- Physical characteristics of the culture vessels.
Most of these conditions are set at the beginning of a plant tissue culture process. However, we can change these conditions in different phases of a plant growth cycle. Apart from these, the chemical environment of the media, the culture media composition, and pH are also set at the beginning of the process. This ensures that the plant cells are always under optimal conditions for nourishment.
As plants perform photosynthesis inside the vessel, they produce gases like carbon dioxide. When there is high CO2 during the light period, it can lead to growth inhibition and even death of tissues. Thus, the aerial microenvironment is dependent on the gas exchange between internal and external environments.
Relative humidity and the air temperature inside the vessel are critical for growth. As the culture medium contains high water content, the relative humidity of the inside air is always higher than that of, for example, a greenhouse. When the relative humidity is above the optimal level, plants cannot efficiently exchange water vapor with the surroundings. This leads to the closure of pores present on the leaves and other plant parts. This can also lead to physiological and morphological disorders in plants such as hyperhydricity, also know as vitrification. To learn more about hyperhydricity and how it affects tissue culture experiments read our dedicated article "Hyperhydricity in tissue culture and methods to deal with it". We will talk about other disorders in detail in upcoming articles!
What can we do to have a better gas exchange? Aeration in culture vessels can be improved by using microporous filter membranes, by using diffusible films, or forced aeration using air pumps. Keeping aerial microenvironment in mind, we have recently developed our new culture vessel model "inVenti⁺ cultivation vessels with filter". Our filter vessel offers flexibility in the amount of required gas exchange. It uses high-quality filters which not only provide protection against microorganisms, but also do not absorb water. This feature facilitates an aerial microenvironment with reduced humidity for plantlets.
This is one of the important microenvironmental factors that control plant growth, development, metabolism, and even chlorophyll content in plant cells/tissues. The light source, light spectrum, and intensity are crucial parameters that influence shoot regeneration, the fresh weight of plantlets, and secondary metabolite biosynthesis during plant tissue culture. One fascinating fact is that light can also impact the efficacy of plant growth regulators in media. It also influences levels of endogenous hormones (hormones produced by plant cells during development).
Though growth hormones play the most important role in determining the development of different plant organs. However, light intensity is another important factor that plays a role in the development of different plant parts. If the light intensity increases, it can lead to rising in temperature which will significantly reduce the growth of plant tissues.
For good growth, light direction is important too. You need to place culture vessels in growth racks/chambers in a way that they are exposed to evenly spread and an adequate amount of light.
Temperature plays an essential role in the growth and development of in vitro plants. You need to make sure that there is a balance and uniformity in heat distribution inside the culture vessel. If the temperature is too high above the optimum, the plantlets will experience fast metabolic activities. This will use up most of the nutrients and water from media faster, hence, plantlets will not show good growth. Low-temperature conditions on the other hand would lead to slowing down of metabolic activities.
Physical properties of culture vessels
Vessels are important for having an efficient workflow for plant tissue culture. They should be designed in such a way to encourage efficient air exchange, reduced humidity, and adequate ethylene levels. The physical properties of the vessels, tubes, and how their lids/caps close affect the interface between the inside and outside environment. Whether the vessels are made of glass, polypropylene, polycarbonate or polystyrene, etc. can also have an impact. Thus, the physical properties of culture vessels have a large influence on the growth microenvironment of the plantlets.
When we talk about vessels, we also need to keep in mind the vessel's ability to allow gas exchange and also transmitting uniform and adequate quality of light to plantlets inside. Different criteria such as preventing contamination and provision of adequate growing area (height and width of the vessel) are also considered while designing and constructing culture vessels.
Optimum utilization of available space is also important for micropropagation facilities. Our "inVenti⁺ cultivation vessels with filter" have shown remarkable results in terms of efficient use of space for plantlet growth. Not only this, they can be efficiently used in growth chambers as their rectangular shape allows for placing vessels next to each other. If you are curious about these vessels, then you can read more about them here!
Now, what if you do not care about these conditions? What will happen then? Let me point out a few potential consequences:
- There could be various types of contaminations;
- There could be differences in rate of growth at each stage;
- It could cost you more to start a new experiment; and
- There might be shortage of plantlets or no plants at all.
Therefore, it is crucial to know about these conditions for your chosen plant species to provide a better microenvironment during the tissue culture process.
We hope this article will be useful for many of you to understand plant tissue culture better. For more articles like this, keep checking our space!
By Nancy Bhatia | 28-July-2021
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