Cryopreservation

Cryopreservation is a conservation method that allows the long-term storage of samples at low temperature up-to -196C making sure that it does not affects cell's viability.

It is an important and efficient method for preservation of world's genetic resources.

It allows long term conservation of biological material

Its simplicity and applicability make it an efficient method.

The plant material stored can be- Seed, Shoot tip, Zygotic embryo, somatic embryo, Pollens, different types of plant cells, tissue and organs.

Various means for storage at low temperature:

At -79°C over solid CO2
At-80°C in Deep Freezers 
At -150°C in Vapor phase
At -196°C in Liquid Nitrogen

These varying temperatures are also called as Sub-Zero temperature.

PRINCIPLE

The Sub-Zero temperature (below0°C) makes the cells suspended due to slow down of biological reactions including metabolism, cellular transport, division etc. The cells remain in this suspended state until the temperature is restored.

This condition is also called as "Zero metabolism" OR "Nondividing state"

But this ultra-low temperature also disrupts or damage the cells by:

• Freezing at such low temperature leads to formation of Ice crystals that damage the cell membrane and disrupt its structure.
• The formation of ice crystals increases osmolyte concentration inside cell that disturbs cellular structure.

This can be overcome by addition of Cryoprotectants.

CRYOPROTECTANTS: To make an ease from above drawbacks of freezing the biological material some chemicals were added to the samples that results in the cellular intactness.

Meaning: Cryoprotectants are the chemical substance can be permeable or non-permeable that leads to lower down the effect of subzero temperature on cryopreserved sample.

The concentration should be optimum.

Examples: Dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol, polyvinyl pyrrolidone, Glycol, Ethylene glycol, Glycerol and sugars etc.

Steps involved in 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

STEP1: Development of sterile tissue cultures

The morphology and physiology of plant material to be selected plays important role in cryopreservation. The cells must be viable.

This step affects the ability of cells to survive during the process.

The cells should be frozen in late lag or exponential phase.

Meristematic cells are also preferable.

STEP2:  Addition of cryoprotectants and pre-treatment

Cryoprotectants were added to minimize the damage caused by freezing and thawing

Cryoprotectants were added during freezing at low temperature and removed during thawing.

• 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 were preferred for more effective cryopreservation.

VITRIFICATION: A process in which the cells achieve glass formation by cooling without ice crystal formation. This can be done when cryoprotectants were make into highly concentrated solitons.

Dehydration:  The cells get dehydrated by cryopreservation this enable them to withstand imersion in liquid nitrogen.

STEP3: Freezing

The rate of freezing can be selected depends on sample.

The sensitivity of the cells to low temperature is variable and largely relies on the plant species.

Different tissue has different sensitivity for cooling are so 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 rate 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.

https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5395684_gr1.jpg

2. Rapid freezing method:

The relationship between dehydration and cooling rate is critical factor in freezing step. As temperature decreases, the intracellular kinetic energy between cells also decreases so loss of water also decreases from the cells. Rapid cooling rates do not allow sufficient time for water to leave before vitrification occurs, thus cellular water content remains high and crystallization is more likely to occur

– 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.

The initial slow freezing in stepwise freezing reduces the intracellular freeze water by dehydration. So, in early ice is formed outside cells .

STEP4: 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 N2nrefrigerator 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.

STEP5: THAWING

Thawing involves warming the frozen biological sample back to its original soft form.

It works best when done quickly.

The ice starts disappearing soon and has less damaging effect.

Main aim is to bring back cells to original condition.

Can be done by retrieval of culture vials from liq nitrogen.

Done by:

STEP6: RECULTURE

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.