Pollen storage is a specialized technique used to preserve the male reproductive material of plants for later use in controlled breeding programs and genetic conservation. Storing pollen overcomes limitations like the short viability window of fresh pollen or the lack of synchronization between parent plants’ flowering times. By storing pollen, researchers can cross species that bloom months or even years apart, or ensure that rare genetic material is available for hybridization. Success relies heavily on carefully controlling the pollen’s moisture content and temperature to halt metabolic activity without causing cellular damage.
Collecting and Preparing Pollen for Storage
The first step in successful pollen banking is collecting the material at the optimal time, which is typically when the anthers dehisce to release the grains. This collection often occurs early in the morning when the flowers first open, though timing is species-dependent. Pollen can be collected by gently tapping the flower over a clean surface, brushing it off the anthers, or using a small vacuum device for larger quantities.
After collection, the most critical preparatory step is desiccation, or drying, to reduce the internal moisture content of the pollen grain. Fresh pollen can contain 20–30% water, but this high level is detrimental for storage, as it encourages metabolic activity and can lead to cell damage if frozen. The goal is to reduce the moisture to a specific level, generally between 5% and 10%.
This desiccation prevents the formation of damaging ice crystals in the cell cytoplasm during freezing and promotes a state of suspended animation. This is achieved by placing the collected pollen in a sealed container with a desiccant, such as silica gel or a saturated salt solution, which creates a controlled, low-humidity environment.
Short-Term Preservation Methods
Once the pollen moisture is stabilized, short-term preservation focuses on slowing down the natural metabolic processes that lead to viability loss. For storage lasting from a few days up to a few months, refrigeration is an effective, simple method. Pollen is typically stored at temperatures ranging from 4°C to 10°C, which significantly extends viability compared to storage at room temperature.
The pollen must be sealed within air-tight containers, such as glass vials or gelatin capsules, to maintain the low moisture content achieved during the drying phase. Even in the refrigerator, exposure to ambient air can lead to the reabsorption of water, which reactivates metabolism and drastically shortens the pollen’s lifespan. Maintaining this low-temperature, low-humidity environment ensures that the pollen’s internal biological clock is slowed, allowing it to remain functional for a limited period.
Long-Term Cryogenic Techniques
For preservation spanning multiple years or even decades, ultra-low temperatures are necessary to completely halt all biological and enzymatic degradation. This long-term storage is achieved using cryogenic techniques. The first level of freezing utilizes ultra-low temperature freezers, typically set to -20°C or -80°C, which can preserve certain desiccation-tolerant pollen types for one to three years. Pollen stored at -80°C retains viability far longer than that stored at warmer freezer temperatures.
The ultimate method for indefinite conservation is cryopreservation in liquid nitrogen (LN), where the temperature reaches -196°C or, in the vapor phase, -150°C. At this temperature, all cellular activity is completely suspended, allowing the pollen to be stored for an undetermined period. The preparatory desiccation is paramount for cryopreservation, as the ultra-low temperatures would cause any remaining free water to form lethal ice crystals inside the cells. While some plant cells require cryoprotectant chemicals to survive this process, mature, desiccation-tolerant pollen is often simply dried and sealed in cryovials before being plunged into the liquid nitrogen. This method is a reliable technique for creating permanent pollen banks for genetic resource conservation.
Assessing Viability and Rehydration
After a period of storage, it is important to assess the pollen’s ability to perform its function before use in a pollination program. Viability testing confirms the stored pollen is still alive and functional. A common technique is the in vitro germination test, where a sample of pollen is placed on a specialized nutrient medium, often a solution containing sucrose, to stimulate the growth of the pollen tube. The percentage of grains that successfully grow a tube provides a direct measure of their fertility.
Another rapid method for assessing viability involves staining techniques, such as the use of Fluorescein Diacetate (FDA). This compound is non-fluorescent until it is taken up by a viable pollen grain and cleaved by internal esterase enzymes, causing the grain to emit a bright yellow-green fluorescence.
Before the stored pollen can be used, it must be rehydrated to a functional moisture level. This process involves gradually exposing the pollen to a humid environment, such as a closed chamber with high relative humidity, for a specific period. Gradual rehydration prevents the cells from bursting due to a rapid influx of water, ensuring the pollen tube can properly emerge and complete fertilization.