Principle of Cryopreservation:
- Cryopreservation is a process of preserving or storing cells, tissues, organs or any other biological materials from any potential damage by maintaining the materials at very low temperature (typically -80 °C using solid CO2 or −196 °C using liquid Nitrogen.
- In cryopreservation, very low temperatures is used to preserve living cells and tissues and maintain their viability. Unprotected freezing is normally lethal.
- Cryopreservation is based on the conversion of water present in the cells from a liquid to a solid state.
- When cooling below 0°C, the biological effects are dominated by the freezing of water, which typically constitutes at least 80% of the tissue mass.
- The cell water requires much lower temperature to freeze (even up to -68°C) due to the presence of salts and organic molecules in the cells, in comparison to the freezing point of pure water (around 0°C).
- The metabolic processes and biological divisions in the cells/tissues are almost stopped when stored at low temperature.
Process of Cryopreservation:
- The cryopreservation of plant cell culture followed by the regeneration of plants involves the following steps:
- 1. Development of sterile tissue cultures
- 2. Addition of cryoprotectants and pre-treatment
- 3. Freezing
- 4. Storage
- 5. Thawing
- 6. Re-culture
- 7. Measurement of viability
- 8. Plant regeneration
The features of the above steps are described as follows:
step I: Development of sterile tissue culture:
- One of the important steps is the selection of plant species with reference to morphological and physiological characters .
- It directly influence the ability of explant to survive cryopreservation.
- Any tissue from a plant can be employed for cryopreservation e.g. meristems, endosperms, embryos, ovules, seeds, cultured plant cells, calluses, protoplasts.
- Out of these, meristematic cells and suspension cell cultures which are in the late lag phase or log phase are most appropriate.
step II: Addition of cryoprotectants and pre-treatment:
- The compounds that can prevent the damage caused to cells by freezing or thawing are called as cryoprotectants.
- Cryoprotectants reduce the freezing point and super-cooling point of water.
- As a result, the ice crystal formation is delayed during the process of cryopreservation.
- Cryoprotectants used are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide.
- Among them, DMSO, sucrose and glycerol are most commonly used.
- Generally, a mixture of cryoprotectants instead of a single one is preferred for more effective cryopreservation without damage to cells/tissues.
step III: Freezing:
- The sensitivity of the cells to low temperature is variable and largely relies on the plant species.
- The different types of freezing methods used are as follows:
- 1. Slow-freezing method:
- The tissue or the essential plant material is allowed to slowly freeze at a slow cooling rates of 0.5-5°C/min from 0°C to -100°C.
- Then it is transferred to liquid nitrogen.
- Slow-freezing method facilitates the flow of water from the cells to the outside.
- This avoids intracellular freezing and promotes extracellular ice formation.
- Because of this, the plant cells are partially dehydrated and can survive better.
- The slow-freezing technique is successfully employed for the cryopreservation of suspension cultures.
- 2. Rapid freezing method:
- This process is quite simple.
- In this technique, the vial containing plant material is plunged into liquid nitrogen.
- During rapid freezing, reduction in temperature from -300° to -1000°C/min occurs.
- The freezing process occurs so quickly that small ice crystals are formed within the cells.
- In addition to it, the growth of intracellular ice crystals is also minimum.
- Rapid freezing technique is applied for the cryopreservation of shoot tips and somatic embryos.
- 3. Stepwise freezing method:
- This technique is a combination of slow and rapid freezing procedures having the advantages of both, and occurs in a stepwise manner.
- Firstly, the plant material is cooled to an intermediate temperature.
- Then it is kept there for about 30 minutes.
- Finally, it is rapidly cooled by plunging it into liquid nitrogen.
- Stepwise freezing method has been successfully applied for cryopreservation of suspension cultures, shoot apices and buds.
- 4. Dry freezing method:
- It has been reported that the non-germinated dry seeds can survive freezing at very low temperature in comparison to water-imbibing seeds which are sensitive to cryogenic injuries.
- In a similar way, dehydrated cells are observed to have a better survival rate after cryopreservation.
step IV: Storage:
- The frozen cultures should be maintained at the specific temperature.
- Generally, the frozen cells/tissues are maintained at temperatures in the range of -70 to -196°C for storage.
- Although, with temperatures above -130°C, ice crystal growth may take place inside the cells which decreases viability of cells.
- The ideal storage is done in liquid N2 refrigerator at 150°C in the vapour phase, or at -196°C in the liquid phase.
- The final aim of storage is to halt all the cellular metabolic activities and preserve their viability.
- The temperature at -196°C in liquid nitrogen is regarded as ideal for long term storage.
- A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is necessary.
- It is essential to check the viability of the germplasm time and again in some samples.
- Proper documentation of the germplasm storage should be done.
step V: Thawing:
- Thawing is usually performed by plunging the frozen samples in ampoules into a warm water (temperature 37-45°C) bath with robust swirling.
- By this process, rapid thawing (at the rate of 500- 750°C min-1) takes place, and this preserves the cells from the damaging effects from ice crystal formation.
- As soon as the thawing occurs (ice completely melts), the ampoules are transferred to a water bath at temperature 20-25°C at the same instant.
- The cells get damaged if left in warm (37-45°C) water bath for long time.
- For the cryopreserved material (cells/tissues) where the water content has been decreased to an optimal level before freezing, the process of thawing becomes less vital.
step VI: Re-culture:
- To remove cryoprotectants, the thawed germplasm is washed various times.
- Following standard procedures, this material is then re-cultured in a fresh medium.
- In some cases, the direct culture of the thawed material is preferred without washing.
- It is so because certain vital substances, released from the cells during freezing, are assumed to enhance in vitro cultures.
step VII: Measurement of viability:
- The measurement of survival or viability of the frozen materials can be performed at any stage of cryopreservation or after thawing or re-culture.
- The techniques used to determine viability of cryopreserved cells are the same as applied for cell cultures.
- The commonly used techniques are staining techniques using triphenyl tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA).
- The entry of cryopreserved cells into cell division and regrowth in culture is the best indicator to measure the viability of them.
- This can be evaluated by the using following expression.
step VIII: Plant regeneration:
- The regeneration of the desired plant is the ultimate purpose of cryopreservation of germplasm.
- The cryopreserved cells/tissues have to be carefully nursed, and grown for appropriate plant growth and regeneration .
- Along with maintenance of proper environmental conditions, addition of certain growth promoting substances is often essential for successful plant regeneration.
Limitations for Cryopreservation:
- An individual with good technical and theoretical knowledge of living plant cells as well as cryopreservation method is required.
Precautions for cryopreservation:
- The formation of ice crystals inside the cells should be prevented as they are responsible for causing injury to the organelles and the cell.
- Cells might be damaged if the intracellular concentration of solutes is high.
- Leakage of certain solutes from the cell during freezing should be checked.
- The physiological status of the plant material is also essential.