Tissue culture (TC) plants are micropropagated plantlets cultivated in a specialized, sterile environment within sealed containers. This method allows for the rapid, large-scale production of genetically identical plants free from pathogens. Storage becomes necessary to manage inventory, delay the planting or “de-flasking” process, or hold plant material for transport. The goal of storage is to maintain the plantlets in a healthy, dormant state within their sterile vessels, preventing them from outgrowing their containers or exhausting their nutrient supply.
Preparing Tissue Cultures for Storage
Successful storage begins with a thorough inspection before the plant material is placed into a reduced-growth environment. Every vessel must be visually checked for any signs of contamination, such as bacterial films, cloudy media, or fuzzy mold growth. Only cultures that appear completely sterile and healthy should proceed to storage, as contamination accelerates under cool conditions.
The plantlets should be a vibrant green color and possess sufficient growth medium, which acts as both their water and nutrient source. Vessels with plantlets that have grown excessively large or those where the agar medium has noticeably shrunk or browned should be set aside for immediate transfer to fresh media or planting out. Ensuring the vessel lids are securely sealed is important, as this seal maintains the sterile environment and prevents desiccation (water loss) during storage.
Optimal Conditions for Short-Term Storage
Short-term storage, often used for periods up to 6 to 12 months, relies on reducing the plantlets’ metabolic rate to conserve resources. This is achieved by lowering the ambient temperature, effectively placing the plantlets into a state of near-dormancy. Most plant species respond well to refrigeration within 4°C to 15°C, with many common varieties thriving near 4°C to 5°C.
Maintaining a steady, consistent temperature is important, as fluctuations can stress the plantlets and disrupt their slowed metabolism. Placing culture vessels in a home refrigerator’s vegetable crisper or a dedicated laboratory incubator is preferable to using areas near cooling vents or refrigerator doors, which experience frequent temperature swings. Tropical varieties often require the upper end of this range to prevent chilling injury.
Light requirements during short-term storage are significantly reduced compared to active growth conditions. While some species benefit from low light intensity (typically between 0.5 and 15 µmol m⁻² s⁻¹) to maintain photosynthetic ability, others can be stored effectively in complete darkness. The low-light regimen helps preserve the plantlets’ photosynthetic apparatus, ensuring they are ready to resume normal growth once removed from storage.
The duration of short-term storage is limited by the finite resources contained within the vessel. The plantlets will deplete the sugar and mineral salts from the agar medium, and water content will slowly decrease through the vessel walls. When the medium dries out or the nutrients are exhausted, the plantlets will begin to show signs of stress and decline, signaling the end of the viable storage period.
Recognizing and Addressing Storage Failure
Storage failure is categorized into two main issues: biological contamination and physiological decline due to resource exhaustion. Contamination, often from airborne fungi or bacteria, is recognized by visible signs like fuzzy growth on the media or plant tissue, or a cloudy, slimy appearance in the agar. If contamination is detected, the affected vessels must be immediately isolated and discarded to prevent spores from spreading.
Physiological decline, caused by the depletion of media resources, manifests as changes in the plantlets’ appearance. Nutrient exhaustion results in chlorosis (yellowing of the leaves due to a lack of chlorophyll), which can progress to tissue death, known as necrosis. For instance, yellowing of older leaves suggests a deficiency in a mobile nutrient like nitrogen, while yellowing of new growth may indicate a lack of an immobile nutrient such as iron.
When plantlets exhibit signs of decline, such as significant yellowing, browning of the media, or stunted growth, they have reached the limit of their storage life. The corrective action is to remove the viable plantlets and transfer them to fresh, sterile growth media in a process called subculturing. This action provides a renewed supply of water and nutrients, allowing the plantlets to recover and resume active growth.