• 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.

Cryopreservation: Principle, Process, limitations and precautions

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• In vitro method is an advanced technology of ex situ conservation for the preservation of genetic materials.
• In vitro methods employing shoots, meristems and embryos are ideally suitable for the preservation of germplasm of vegetatively propagated plants.
• This approach can also preserve plants with recalcitrant seeds and genetically engineered materials along with orthodox plants.
• The conservation implies preservation of cells, calluses or tissues of selected plant species in sealed test tubes in in vitro method.
• This conservation depends on principle that plant cells are totipotent and plant materials can be kept alive for infinite period of time as in vitro cultures.
• Advantages:
  • Requires less area for preservation of large quantities of materials.
  • The germplasm are preserved in pathogen-free environment.
  • Genetic materials are protected against the nature’s hazards.
  • Large number of plants can be obtained from the germplasm stock whenever required.
  • Since the germplasm is kept under aseptic conditions, it can be easily transported.
• Disadvantage:
  • It requires constant electricity, skilled manpower and high technology.

Techniques for the in vitro conservation of germplasm:

1. Cryopreservation (freeze-preservation)
2. Cold storage
3. Low-pressure and low-oxygen storage

1. Cryopreservation:

• Cryopreservation (Greek, krayos-frost) actually means preservation in the frozen state.
• Cryopreservation is based on the principle where the metabolism or division of plant cells and tissue cultures are completely halted by reducing temperatures with the involvement of cryoprotectants.
• This approach is highly applicable for the conservation of plant species in danger of extinction.
• Cryopreservation broadly refers to the storage of germplasm at very low temperatures:
  • i. Over solid CO₂  (at -79°C)
  • ii. Low temperature deep freezers (at -80°C)
  • iii. In vapour phase nitrogen (at -150°C)
  • iv. In liquid N₂ (at -196°C)
• Out of these, the most commonly used cryopreservation is use of liquid nitrogen.
•  At the temperature of liquid nitrogen (-196°C), the cells remain in totally inactive state and thus can be preserved for long time.
• Cryopreservation has been applied for germplasm conservation of several plant species e.g. rice, wheat, peanut, cassava, sugarcane, strawberry, coconut.
•  Various plants can be regenerated from cells, meristems and embryos stored in cryopreservation.

2. Cold Storage:

• Germplasm conservation at a low and non-freezing temperatures (1-9°C) is involved in this stage.
• In contrast to complete stoppage of cryopreservation, the growth of the plant material is slowed down in cold storage.
• Thus, cold storage is considered as a slow growth germplasm conservation method.
• This approach avoids the cryogenic injuries of plant material.
• This step is simple, economical and results germplasm with good survival rate.
• This approach stores many in vitro developed shoots/plants of fruit tree species for e.g. grape plants, strawberry plants.
• With the addition of a few drops of medium periodically (once in 2-3 months), virus- free strawberry plants could be conserved at 10°C for about 6 years.

3. Low-Pressure and Low-Oxygen Storage:

• Low-pressure storage (LPS) and low-oxygen storage (LOS) are other alternatives for the cryopreservation and cold storage which have been developed for germplasm conservation.

Low-Pressure Storage (LPS):

• The atmospheric pressure surrounding the plant material is decreased in low pressure storage.
• This yields in a partial reduction of the pressure exerted by the gases around the germplasm.
• The lowering of partial pressure decreases the in vitro growth of plants (of organized or unorganized tissues).
• Low-pressure storage systems are essential for both short and long-term storage of plant materials.
• The short-term storage is specifically useful to enhance the shelf life of many plant materials e.g. fruits, vegetables, cut flowers, plant cuttings.
• The storage of germplasm grown in cultures can be done for long term under low pressure.
• Besides germplasm preservation, LPS decreases the activity of pathogenic organisms and prevents spore germination in the plant culture systems.

Low-Oxygen Storage (LOS):

• In the low-oxygen storage, the oxygen concentration is decreased, but the atmospheric pressure (260 mm Hg) is maintained by the addition of inert gases (specifically nitrogen).
• There is reduction in plant tissue growth if the partial pressure of oxygen is below 50 mm Hg.
• It is because, with less availability of O₂, the production of CO₂ is low.
•  As a result, the photosynthetic activity is decreased, thereby halting the plant tissue growth and dimension.
• Limitations:
  • The long-term conservation of plant materials by low-oxygen storage may halt the plant growth after certain dimensions.

In vitro methods of germplasm conservation

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