Cryopreservation

cryopreservation and its steps, principles, significance, cryoprotectants

1. CRYOPRESERVATION PRESENTED TO : Dr . LAL AHAMED Associate Professor Genetics & Plant Breeding PRESENTED BY S RAVI TEJA BAM 20-23

2. INTRODUCTION Cryopreservation is the process which refers to the “ preservation in the frozen state”. It is derived from the Greek word ‘KRYOS’ meaning ‘FROST’. The term “cryopreservation” means storage at very low temperature such as in deep freezers (-80°C) in vapor phase nitrogen (-150°C) or in liquid nitrogen (-196°C). Cryopreservation refers to the storage of cells, tissues & organs at the ultra- low temperature of liquid nitrogen. At such low temperatures, the stored material enters in a state of “absolute quiescence” as all the physical & the biochemical reactions are practically halted. Thus, cryopreservation is a long term storage techniques with very low temperatures to preserve the structurally intact living cells & tissues for extended period of time at a relatively low cost. The science pertaining this activity is known as “CRYOBIOLOGY”.

3. 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.

4. The first successes in plant cell cryopreservation were achieved in the early 1970s. Cell suspension cultures have been among the most amenable materials to this method of preservation. The procedure for these materials involves the following stages: pre-growth, cryoprotection, cooling (protective dehydration), storage, increasing the temperature, post-thaw treatment and recovery growth. Cryopreserved embryogenic suspension cultures from which plants can be regenerated are a potentially valuable tool for conservation. Because of concerns regarding genetic stability, most cryopreservation research for genetic conservation has concentrated on organized cultures such as shoot tips, shoots and zygotic embryos.

5. 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

6. SELECTION OF MATERIAL Material chosen for cryopreservation should be far as possible in meristematic state. For selecting a material, a number of facts are taken into account: 1. The nature of cells. 2. The density of cells in the vials to be preserved. Cell cultures are generally preserved in lag or early exponential phase of growth. Young, highly cytoplasmic & small cells which are non vacuolated & in small aggregates are good materials to be selected for cryopreservation. In some species, it may be important to use highly embryonic cell cultures since non- embryonic or poorly embryogenic cultures show poor or no regrowth after thawing.

7. 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.

8. ADDITION OF CRYOPROTECTANTS

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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 N 2  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.

15. 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.

16. 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.

17. 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.

18. 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.

19. Recalcitrant seeds present a dual problem in cryopreservation: they are often large and therefore prone to structural injury and they are very sensitive to dehydration. Perhaps the most promising and intriguing of the new developments in cryopreservation involves artificial seed technology. Dereuddre et al. (1991) pioneered a technique involving encapsulation, dehydration and cryopreservation of shoot tips or somatic embryos. These are encapsulated in an alginate gel, dehydrated in air or by incubation in a hypertonic sucrose solution and cooled rapidly. This can give much higher survival levels and result in less structural damage than conventional approaches.

20. Some plant materials have survived well under conventional cryopreservation while others have remained completely unresponsive. There is a large group of materials that survive to some degree at the cell and tissue level, but with some physical damage. In these cases callusing, which has the attendant risks of somaclonal variation, is employed instead. Recent efforts have increased the number of species that can be cryopreserved as shoots and have improved the quality of cryopreserved specimens. However, progress has been slow and has involved a relatively narrow range of species.

21. Cryopreservation is the key to conserving many crops we rely on for our food and agriculture. While most of the world’s food plants, such as rice, maize and wheat, can be conserved as seeds in gene banks, the same cannot be said for some other important crops.

22. SIGNIFICANCE OF CRYOPRESERVATION. There are various advantages of this technique. These are as follows: 1. Cryopreservation of gametes, embryos etc. prevents genetic drift. 2. It safeguards genetic integrity of valuable stains. 3. It offers generation time & allows further contribution of genetics. 4. It eases transportation of genetic stock. 5. It causes decrease of disease transmission

23. 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.