The in vitro preservation of plant materials at- 196ºC for a long time using liquid nitrogen is called cryopreservation. Cell suspensions, hybrid protoplasts, pollen grains, seeds and meristem of desired plant races are stored in this medium.

Liquid nitrogen maintains low temperature for the long-term storage. The low temperature reduces the growth rate of the stored cells and delays ageing. Therefore, the plant materials do not lose their viability for a long time.

Plants can be regenerated from the cryopreserved plant materials after a long period of in vitro storage.

Further, the plant materials do not lose their original characters during cryopreservation.
Callus tissues of potato, pea, coconut, rice, wheat, oil palm, strawberry, sugarcane, etc are stored by cryopreservation.

The essential steps of cryopreservation of plant materials are as follows:
• Plant cell suspensions, protoplasts, pollen grains, embryos and bud meristems are generally used for cryopreservation. Cell suspensions and protoplasts are isolated from plants to be cryopreserved. • The embryos, meristem and pollen are collected from proper plant sources.
• The cell suspension contains a large proportion of nutrient medium. The excess medium is removed after sedimentation of cells by keeping the culture in ice-path.

Cryoprotective agents are chemicals which protect plant materials from damaging effects of cold storage. They are often named cryogens. Dimethylsulfoxide (DMSO), Propylene glycol, polyethylene glycol (PEG), polyethylene oxide (PEO), hexamethylene tetramine glucose, Dimethyl acetamate, etc. are important cryogens being used in storage of plant and animal materials.

Plant cells require proper pre-cooling treatment before long-term cold storage. It prevents the formation of intra-cellular ice-crystals in the cells.

During pre-freezing, temperature is lowered gradually to the rate of 10ºC for every 3 minutes. In such a way temperature is reduced upto-135ºC. At this temperature, almost all fractions of freezable water ooze out of the cell. So the cells will not be affected during long term cold storage.

Freezing units like LR-33 Biological freezer-6, Programmed freezer R201 and Mini-freezer R202 are being employed for the pre-freezing treatment.

After pre-freezing treatment, the cell culture is transferred to a small flask or beaker. The flask is insulated with a thick plastic cover to prevent damages by liquid nitrogen. The culture flask is then kept in liquid nitrogen in a container. The container is kept in a refrigerator.

The liquid nitrogen keeps the temperature -196ºC inside the culture vessel. At this temperature there is no metabolic activity in plant cells due to inactivation of cellular enzymes. So the cells remain as such for a long time without any considerable change. By using this method plant materials can be stored for 5 years or more.

Reuse of preserved tissue:

After a sufficient period of cold storage, the culture flask is taken out of the liquid nitrogen and heated gently. The heat is raised slowly but gradually, up to 35-40ºC. This temperature treatment is called thawing.

Quick heating cause dehydration of cells and it results in death of the cells. So sudden heat is avoided. Thawing melts extracellular ice-crystals formed during the long term storage. Immediately after melting of ice crystals the culture flask is kept in a water- bath.
The stored plant cells have cryogen molecules on the surface of the cell walls. The cryogen interferes with growth of the cells into callus and plant regeneration. So cryogens are washed out of the cells and rinsing the cells with distilled water. The water is then centrifuged out to get a pellet of plant cells. These cells are separated from the centrifuge tube and subjected to viability tests.

The viable cells are transferred to a nutrient medium containing auxin and incubated 25ºC for callus induction.
The cultured callus tissues are grown into plantlet in vitro using proper culture media. Then the plants are planted in pots in a green house for proper acclimatization.

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