What is acclimatization in plant tissue culture?

Did you know that many tissue cultured plants do not survive in greenhouse conditions?

The greenhouses or fields have relatively lower relative humidity, higher light level, and an environment full of pests and microorganisms around. These conditions pose extreme stress to plants cultured under in vitro environments. Therefore, a successful tissue culture protocol for a plant species also involves the efficient transfer of plantlets from culture vessels to the ex vitro (field/greenhouse) conditions.

In the tissue culture process, you also need to care about the mother plants which you use for obtaining explants. As much as acclimatization is important for a successful result, taking precautions to prevent contaminations at the beginning of the process is also crucial. You can read more about maintaining healthy mother plants in our article on "How to select and prepare stock plants?".

For now, let us discuss the acclimatization process:

What is acclimatization?

Acclimatization here simply means the adaptation of plantlets to a new environment. Plantlets or shoots in the culture vessels are accustomed to a different micro-environment. You customize this micro-environment in a way that the developing plants experience minimal stress and optimum conditions to grow and multiply. When you transfer these plantlets from the laboratory conditions to the soil, you expose them to different abiotic and biotic stresses. Hence, you need to develop a stepwise acclimatization protocol for your plants to adapt to the natural environment.

But, why do you need a stepwise process for acclimatization?

In the culture vessels, your plantlets are growing in controlled conditions, such as:

• Low level of light;
• High level of humidity;
• Aseptic conditions; and
• Nutrient medium with substantial amount of sugar.

Plants growing in such conditions cannot survive the environmental conditions when they are directly transferred in the open. Because of this reason, the stage of acclimatization is a major bottleneck in the micropropagation of many plant species and requires a carefully curated stepwise process to adapt to an open environment.

What are the problems faced by in vitro plants during acclimatization?

According to published scientific papers, tissue cultured plants experience high mortality during or following transfer to the natural environment. Let us briefly discuss some of the problems that plants grown in tissue culture face during acclimatization:

• The presence of high humidity in the culture vessels leads to abnormal functioning of stomata upon transfer to soil. This directly influences the rate of transpiration in plants or in other words, it affects the rate at which a plant absorbs and utilizes water for growth.
• Due to low light intensity, low carbon dioxide concentration, and high sugar in the media, plantlets are accustomed to a relatively low rate of photosynthesis, which further leads to slow growth.
• As plantlets grow in sterile media, they possess weaker roots when compared with field plants. These weak roots experience difficulty in uptaking nutrients from the soil.
• When plantlets grow in excess amount of plant growth regulators, they show abnormalities in morphology upon transfer to greenhouse conditions.
• When plantlets are transferred from low light intensity and temperature to broad spectrum sunlight and higher fluctuating temperature, then they experience charring of leaves (burning of leaves/browning of plant tissues) and wilting of plants.
• Direct transfer of plantlets to sunlight can also inhibit the process of photosynthesis which is necessary for energy production.
• Another major cause of mortality in tissue cultured plantlets is their sudden exposure to microflora present in the soil. As these plants are not sufficiently resistant to such organisms, they get infected easily and do not survive.

Hence, plants cultivated in vitro are different from field-grown plants and require more attention while adapting to a new environment.

How can you acclimatize your plants?

Let's discuss the measures you can take to acclimatize your plantlets. Also, what factors do you need to keep in mind to ensure the survival of plantlets?

• First, it is necessary to transfer the plants from a tissue culture environment to a greenhouse providing the same environment in which it was cultured in the lab.
• You need to transfer the plantlets to a potting mix, irrigated with inorganic nutrient solution. A variety of potting mixes are available nowadays including peat, vermiculite, soil, sand, etc.
• You can also keep the culture vessels with loose lids for a few days in the greenhouse.
• You can leave the plantlets in shade for 3-6 days under diffused natural light. This would help them to establish under new environment.
• According to scientists, acclimatization should occur in 2-3 phases where you gradually expose plantlets first to greenhouse and then to field conditions.
• In order to adapt plants from high to low humidity, you need to keep them in shade with loose plugs for a week or two. Then you should transfer them to pots containing sterile soil and sand mixture. You can cover these pots with polybags.
• You can also precondition your rooted plantlets in different sucrose solutions (20- 30g/L of concentrations) before transferring to a potting mixture. This would increase the growth of shoots.
• In several studies, it is also suggested to add anti-transpirants like 'paclobutrazol' in the rooting medium. This helps plantlets to have normal funtioning of stomata, thickened roots and also reduces wilting.
• You need to customize these steps for each plant variety based on the plant's growth requirements.

The steps mentioned in this section will shock your plantlets at first as the growth media will change from inorganic media to organic soil. But, this will also activate the photosynthetic activity of the plantlets and prepare them to withstand the natural environment.

By keeping these steps in mind, you can ensure a good start for your plantlets in field conditions, be it for a small or a large-scale production.

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 | 14-September-2021

References

• Hazarika, B. (2003). Acclimatization of tissue-cultured plants. Current Science,85(12), 1704-1712. Retrieved September 13, 2021, from http://www.jstor.org/stable/24109975
• Debergh, P. C. (1991). Acclimatization Techniques Of Plants From In Vitro. Acta Horticulturae, (289), 291–300. doi:10.17660/actahortic.1991.289.77
• Chandra, S., Bandopadhyay, R., Kumar, V., & Chandra, R. (2010). Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnology Letters, 32(9), 1199–1205. doi:10.1007/s10529-010-0290-0
• Kyte, Kleyn, et al (2013) Plants from test tubes: An introduction to micropropagation. Timber press, Inc.
• Bhojwani, S.S., & Dantu, P.K. (2013). Plant Tissue Culture: An Introductory Text. Springer India