How to Prepare Plants for Acclimatization at the Rooting Stage
5 Aug 2021

How to Prepare Plants for Acclimatization at the Rooting Stage

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.

Anjali Singh, MS
Table of Contents

The Basics

Every stage in tissue culture is crucial, starting from establishing the culture, multiplication, rooting, and acclimatization. Gradually adapting the plants to their natural condition is as important as taking care of them at growing stages inside the lab.

The two options that you have are: 

  • Plants nicely adapt to the natural environment
  • They die!

That’s why the transplantation stage is considered to be a major bottleneck in the micropropagation of plants.

Plants grown inside the labs are heterotrophic because of the artificial condition created for their growth. The plants inside the jar receive:

  • Low level of light.
  • Growth medium with sufficient sugar and nutrients amount.
  • Complete aseptic condition to prevent any biotic stress during plant’s growth for the enhanced output.
  • An atmosphere with a low level of humidity.

All these conditions induce a phenotype suitable for the heterotrophic nature of plants. And, these conditions do not support the growth of in vitro plants when directly placed outside in the natural environment. There’s a step-by-step procedure and factors to consider on how to do it!

The precautions and preparations to transfer the plant for acclimatization begin at the rooting stage (Stage-3). This article brings you the perfect process and factor required to be learned for acclimatization of plants, starting from the rooting stage.


Preference Center

Preparing a plant for transplant at the rooting stage

The plants grown in an artificial environment have different phenotypic characteristics than those grown in natural environments. The cultured plants have no functional stomata, weak root system, and poorly developed cuticle leading to more water loss.

Weak or damaged roots of the plants result in the death of plants. That’s why it’s essential to try and focus on inducing proper development of roots in the cultured plants.

But what should you really be concerned about?

Given below are some points that you should do or avoid while rooting:

Don’t use stiffer agar (higher concentration of agar than suggested normally)

The stiff agar will have reduced moisture and will not let the roots develop well. Furthermore, when you will be taking out the plant for the next stage, agar will be badly stuck to the plant that will make it difficult to separate them from roots. Difficulty separating it might break off some roots or damage them.

Moreover, plants don’t get enough nutrients in hard agar because of their poor distribution and uniformity in terms of nutrients.

Now, you might think, how about liquid agar? It can easily allow roots to develop! But this will not be the case. In liquid agar, more fine root hair will develop that will be chunky and firm. And, these roots might make it difficult for the plant to adapt to the normal or natural environment.

So, always go for the normal concentration of agar and your plants’ roots will happily grow!

Use best hormones and ratio of hormones for organ development

Here, the “best hormones” denote the type of hormone best suited for the growth of your plant (You can refer to the literature to search what works best for your plant). For example, in Philodendron it has been observed that 80% of plants survive during transplantation when 2iP or kinetin is used instead of BA in Stage II.

The best cytokinin and auxin ratio leads to the perfect development or regeneration of plant organs. You should know that at stage II the growth media is rich in cytokinin for shoot development, and in stage III, auxin-rich culture media are utilized for root development. And, the amount and type of cytokine you use at stage II will also determine the plant’s responses at further stages.

The third stage might not contain cytokinin in any amount or be added in a small concentration, with increased auxin levels, in the culture media for root development. It has been also observed that decreasing salt levels in the culture media might induce proper development of roots. Furthermore, reducing the concentration of sugar at stage III may encourage photosynthesis in plants that will help the plants at their acclimatization stage.

Related: The 6 most common problems in tissue culture 

Maintaining humidity

Well, here comes the factor of humidity!

Humidity can be maintained as required by proper choice and adjustment into the container’s cover. What does this mean?

You can start preparing your plants for fitting into the natural conditions at stage III only. When a few days are left for the plants to acclimatize, you can lose the cap of the containers to allow water vapor to escape from the culture vessels. For example, the Dianthus plant culture was left uncapped for nine days with 50-70% humidity and that increased the survival of plants to 90%.

But, this also depends on how clean your culture room is, and what percentage of humidity you have been maintaining for your plants. Moreover, if you leave your cultures uncapped for many more days, then, they can build up contaminants inside the vessel, leading to the death of your cultures. So, carefully follow the steps with your cultures.

It’s recommended that lids or caps present on the container are gradually removed and not completely at once! It may shock your plants and cause them stress that can probably lead to their death. You can follow this step in the next stage of acclimatization or transplantation as well!

The other way for acclimation at the rooting stage is to use containers having lids with filters. The filters allow gas exchange between the plant and its atmosphere. Moreover, in some cases, bottom cooling has worked best to reduce the relative humidity in container’s empty spaces.

Do all this while keeping in mind the aseptic condition in your lab and room humidity!

These are some effective ways that you can prepare your plant for acclimatization at its rooting stage. If you are facing any issues in your tissue culture process, you can opt for our consultation services to get all your solutions right away! Visit plant cell technology to learn more about the service, its benefits, and prices.

And, stay tuned to the plant cell technology blogs for innovative content every week!

Happy culturing!!

Source: Giphy


  1. Kyte Lydiane, Kleyn G. John, Scoggins Holly, and Bridgen Mark (2013). Plants from Test Tubes: An Introduction to Micropropagation, 4th ed. Timber Press, Portland, London.
  2. Hazarika, B. (2003). Acclimatization of tissue-cultured plants. Current Science, 85(12), 1704-1712. Retrieved July 29, 2021, from
  3. 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.

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