Tissue Culture Acclimation Guide

Congratulations on purchasing an RPF Labs tissue culture plantlet! This guide will take you through the acclimation process, ensuring you get the best possible start for your young plants.

What is Plant Tissue Culture Acclimation?

Tissue culture acclimation, or hardening, is the process of gradually adapting lab-grown plantlets to more variable, less controlled environments. This is an essential step as plants grown in vitro under sterile and controlled conditions are not used to natural conditions such as light, wind, or pests.

Step-by-Step Guide

Step 1: Unpacking and Clean-Up

Unpack your plantlets from their shipping material with clean hands or sterilized gloves, being very gentle to avoid causing any damage to the delicate plantlets. Place the plantlets on a clean and sterilized work surface.

It's essential to remove any dead or dying tissue, as it can become a breeding ground for pathogens that could harm your plant. Using a clean, sterilized tool (like a pair of fine tweezers or small scissors), trim away any yellowed, brown, or otherwise unhealthy-looking tissue.

Make a lukewarm water bath (RO or distilled water preferred) and rinse off all the agar (gel media). It’s essential to clean off all the agar as this will cause rot after planting.

Before planting, it can be beneficial to treat the plantlets with an anti-fungal solution. This solution can be prepared by mixing a suitable fungicide (like Physan 20 or Captan) with water as per the manufacturer's instructions. Immerse the plantlets in this solution for about 5-10 minutes, and then rinse them gently under sterilized water. This can help prevent the onset of fungal infections during the acclimation process.

Step 2: Initial Planting

After cleaning and treating your plantlets, it's time for planting. Choose a sterile medium suitable for your plant species. This could be sphagnum moss, peat moss (loose or in plug form), perlite, vermiculite, or a mixture of these. The medium should be moist, not wet, as waterlogging can lead to root rot.

Create a small hole in the medium, place the plantlet inside, and gently cover the roots with the medium, leaving the shoot exposed. 

Many plants, especially carnivorous ones, do not have significant roots in TC. Roots can also be black in color which does not indicate root rot. Gently place these TC plantlets in your medium of choice while keeping them upright.

After planting, the next crucial step is covering your plantlets to create a mini greenhouse effect. This can be done using a clear plastic dome, propagation box or bag that is large enough to cover the plant and its pot without touching the leaves. This setup will trap moisture and maintain a high level of humidity around the plant, which is crucial for tissue cultured plants as they transition from a high-humidity lab environment.

To avoid overheating and to let in light, ensure your cover is transparent. Ventilation holes are not needed initially, as the aim is to keep the environment as humid as possible. These can be added or opened later as you start to gradually decrease the humidity levels in the acclimation process.

Remember, the transition from tissue culture flask to the outside world can be stressful for the plantlets, and maintaining a high humidity environment initially can significantly improve the survival rate during this crucial phase.

Step 3: The Hardening Process

During this phase, your plantlets will get used to the conditions they'll face outside of the lab.

Light

Initially, keep the plants in indirect sunlight or under a low intensity grow light. Gradually increase light exposure over a few weeks.

Humidity

High humidity (80-90%) should be maintained for the first week or two.  Gradually reduce the humidity by making small holes in the plastic cover or opening the bag a little every few days.

Temperature

Room temperature, around 20-25°C (68-77°F), is ideal for most plantlets during acclimation.

Watering

Keep the growing medium moist but not wet. It's best to water using a mist spray. As your plant acclimates, gradually reduce the frequency of watering.

Air movement

To help strengthen plant stems, introduce a light fan breeze after a few weeks of the hardening process.

Step 4: Transplanting

Once the plantlets are hardened off, usually after 4-8 weeks, they can be transplanted to bigger pots with regular potting soil.

Step 5: Continued Care

Monitor your plants carefully for signs of stress, such as wilting or discoloration. Continue to keep conditions stable, avoid overwatering, and slowly introduce the plants to their final growing conditions.

Troubleshooting

Plantlets are wilting. This could be due to low humidity or overexposure to light. Increase humidity or decrease light intensity.

Leaves turning yellow or brown: This could be due to overwatering or nutrient deficiency. Make sure the medium is not waterlogged and consider applying a mild fertilizer.

Slow growth or no growth: Check the light and temperature conditions. The plantlets might need more light or a warmer environment.

Remember, patience is key. Acclimating tissue culture plantlets can take several weeks, but with careful attention, you'll see your plantlets transform into sturdy, healthy plants. If you have any further questions or concerns, feel free to contact us. Happy gardening!

12/19/24 Debora Sweeney