As plantlets from tissue culture are tender and can easily be damaged by abrupt changes in their surroundings, the hardening process for plant tissue culture involves transitioning plantlets from the controlled, sterile, and more humid environment of a culture vessel to the more variable conditions of the outside environment.

 

Objective: To acclimatize in vitro-raised plantlets to external environmental conditions before transplanting them to soil.

Scope: This procedure is applicable to plantlets grown via tissue culture that are ready for hardening.

 

 

 

 

PRECAUTIONS

Handle plantlets gently to avoid physical damage.

Always use sterilized tools and equipment to prevent contamination.

Do not overcrowd the trays or pots; ensure proper spacing for healthy growth.

Use personal protective equipment (gloves, lab coat) during the handling process.

 

 

PREPARATION

Ensure the hardening area is clean and free from pests and diseases.

Prepare trays or pots filled with a sterile potting mix or cocopeat.

Plantlets are carefully taken out from the culture vessels.

Residual medium or agar adhering to the roots is gently rinsed off with sterile distilled water.

Plantlets are initially potted in small containers filled with a sterilized mixture, often consisting of peat moss, vermiculite, or perlite. These substrates are generally low in nutrients.

 

 

 

INITIAL ENVIRONMENT

Nutrients: The plantlets have limited ability to take up and process nutrients. Therefore, it’s crucial to provide a diluted nutrient solution in the beginning.

Lighting: Keep the plantlets under diffused light (about 1000-2000 lux) for the first 3-4 days.

Humidity: High humidity (around 90-95%) should be maintained initially and then gradually decreased over time to match ambient levels over the next 2-4 weeks.

Temperature: Ensure the temperature is in the range of 25-28°C.

 

 

 

SCHEDULE

First Week: Use a nutrient solution that’s 25% of the full strength. This means if you have a standard fertilizer recommendation, only use 25% of that amount. Keep humidity at 90-95%.

Second Week: Increase the nutrient solution to 50% of the full strength. Lower humidity to 80-90%.

Third Week: Further increase the nutrient solution to 75% of the full strength. Lower humidity to 65-75%.

Fourth Week and Onward: By now, the plantlets should be acclimated enough to handle full-strength nutrient solutions. Acclimated plantlets should be strong enough to handle exposure to ambient humidity levels around 50-65%.

 

MONITORING & CARE

Regularly check the plantlets for signs of nutrient deficiencies or toxicities. Yellowing leaves might suggest a nutrient imbalance.

Monitor the moisture level in the substrate. While it should be kept moist, it shouldn’t be waterlogged.

Look out for signs of pest or disease infestations. Tissue cultured plantlets can be more susceptible initially due to their tender nature. If any are observed, take appropriate measures immediately.

 

 

FINAL TRANSFER

After 2-4 weeks (or longer, depending on the plant species) of the hardening process, the plantlets should be robust enough to be transferred to regular soil or a growing medium in either pots or directly in the field.

Once the plantlets have established a robust root system, have acclimatized to ambient humidity, and can withstand full ambient light, they are ready for transfer to your medium of choice. We recommend soil for genetic preservation.

 

POINTS TO REMEMBER

The nutrient strengths mentioned are general guidelines. Different plant species might have specific requirements.

The transition from tissue culture media to the outside environment is a stressful period for the plantlets. The aim of the hardening process, including adjusting nutrient strengths, is to make this transition as smooth as possible.

Always monitor the plants closely and adjust conditions based on their response. Over time, with experience, you’ll develop a better sense of what each specific plant species requires for successful acclimatization.