Plant Cryopreservation

Plant Cryopreservation. Now that in the past few blogs we have studied how to make somatic hybrids let us now study how to preserve them. you see plant tissue or cell preservation is a very important and sensitive procedure because we never know what will happen in the near future whether a disease might spread in particular species of plant, it might go extinct, or the novel hybrids we have produced we might lose their genetic composition and won't be able to regenerate. So in order to prevent all this cryopreserving, the plant specimen is important.

 Cryopreservation

The principle involved in cryopreservation is to bring a plant cell and tissue cultures to a zero metabolism or non-dividing state by reducing the temperature in the presence of cryoprotectants. Cryopreservation simply means the storage of germplasm at very low temperatures. The most commonly used cryopreservation is by employing liquid nitrogen (-1 96'C), the cells stay in a completely inactive state and thus can be conserved for long periods.

Cryopreservation of plant cells is a process of preserving cells at very low temperatures i.e., 77K or −196°C. At low temperatures, any enzymatic or chemical activity which might cause damage to the biological material in question is effectively stopped and the cells remain in a completely inactive state.

Precautions to be taken:-

1. Formation of ice crystals inside the cells should be prevented as they cause injury to the organelles and to the cell.

2. High intracellular concentration of solutes may also damage cells.

3. Sometimes, certain solutes from the cell may leak out during freezing procedure. Cryoprotectants also affect viability of cells.

4. The physiological status of the plant material is also important.

Cryopreservation of plant cell culture followed by the regeneration of plants involves the following stages:-

1. Development of sterile tissue cultures
2. Addition of cryoprotectants and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration

 There are many tissue from a plant which can be used for cryopreservation e.g. meristems, embryos, ovules, seeds, cultured plant cells, protoplasts, calluses. Among all of these, meristematic cells and suspension cell cultures, in the late lag phase or log phase are most suitable.

What are cryoprotectants?

Cryoprotectants are the compounds that can prevent the damage caused to cells by freezing or thawing procedure. Because of it the ice crystal formation is retarded during the process of cryopreservation. Some of the cryoprotectants that are used are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, and sucrose. From all of these, DMSO, sucrose and glycerol are most widely used.

Freezing = The sensitivity of different cells to low temperature is variable and largely depends on the plant species. Three different types of freezing methods are being used.

• Slow-freezing method : In this method the tissue or the requisite plant material is slowly frozen at a slow cooling rates of 0.5-5°C/min from 0°C to -100°C, and then transferred to liquid nitrogen.
• Advantage= During slow freezing some amount of water flows from the cells to the outside. This promotes in extracellular ice formation rather than intracellular freezing. Because of which, the plant cells are partially dehydrated and survive better.
• Rapid freezing method: This technique is quite simple and it involves the plunging of the vial containing plant material into liquid nitrogen. During rapid freezing, a decrease in temperature from about -300 to -1000°C/min occurs. The freezing process is carried out so quickly that small ice crystals are formed inside the cells. This technique is used for the cryopreservation of shoot tips and somatic embryos.
• Stepwise freezing method: This is a combination of slow and rapid freezing procedures (with the advantages of both), and is carried out is a stepwise manner. The plant material is first cooled to an intermediate temperature and is maintained there for about 30 minutes and then rapidly cooled by plunging it into liquid nitrogen.

Storage= Maintenance of the frozen cultures at the specific temperature is same as important as freezing. ln general, the frozen cells are kept for storage at temperatures in the range of -70 to -196°C. Storage is ideally done in liquid nitrogen refrigerator at around -150°C in the vapour phase, or at -196°C in the liquid phase. The ultimate objective of storage is to stop all the cellular metabolic activities and maintain there by maintaining their viability. For long term storage, temperature at -196°C in liquid nitrogen is ideal condition.

Thawing= Thawing is usually carried out by plunging the frozen samples in ampoules into a warm water (temperature 37-45°C) bath with vigorous swirling for few minutes. This method protects the cells from the damaging effects ice crystal formation. As the thawing occurs (ice completely melts) the ampoules are quickly transferred to a water bath at temperature 2O-25°C.

Reculture= After being stored now when the time come we need to reculture them. The thawed germplasm is washed several times to remove cryoprotectants. This material is then recultured in a fresh medium following standard procedure.

Measurement of survival/viability= The techniques employed to determine viability of cryopreserved cells are the same as used for cell cultures. Staining technique such as, Evan's blue and fluorescein diacetate (FDA) are commonly used. The best indicator to measure the viability of cryopreserved cells is to check their entry into cell division.

Plant regeneration= The ultimate purpose of cryopreservation is to regenerate the desired plant. Addition of certain growth promoting factors, besides maintenance of appropriate environmental conditions is often necessary.

Applicaton of Cryopreservation of plant

1. Plant materials can be cryopreserved and maintained for several years, in contrast to an in vitro cell line maintenance which has to be subcultured and transferred periodically to extend viability. Thus, germplasm storage is an ideal method to avoid subculturing, and maintain cells/ tissues in a viable state for many year.
2. Cryopreservation is an ideal method for the long term conservation of cell cultures which produce secondary metabolites (e.g. medicines).
3. Disease (pathogen)-free plant materials can be frozen, and be propagated whenever required.
4. Plant materials from endangered species can be conserved for long time.
5. Conservation of pollen for enhancing longevity.
6. Rare germplasms developed through somatic hybridization and other genetic manipulations can be stored without being lost.
7. Establishment of germplasm banks can be done for exchange of information at the international level.

LIMITATIONS OF GERMPLASM STORAGE

One of the major limitations of germplasm storage are the expensive equipment and the trained personnel.

Demerits of Cryopreservation of plant

1. Does not work efficiently for all strains.
2. Formation of ice crystals inside the cells cause injury to the organelles and the cell.
3. High intracellular concentration of solutes can be very damaging to cells.
4. Sometimes, certain solutes from the cell may leak out during freezing.
5. Cryoprotectants also affect the viability of cells.
6. Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cryopreserved cell can cause damage directly.
7. The physiological status of the plant material is also important.
8. Cryopreservation protocols for many plant species are not available.
9. Though cryopreservation technique is very simple, limited knowledge make this technique highly complex.

Conclusion 

Due to all the above reasons and for conserving the plant materials/ novel hybrids/ endangered plant species and maintaining all the plant-related data in big germplasm banks, the cryopreservation technique is being used. With help of this technique, it helps us to study the plant tissue culture because we only use the cryopreserved plant materials for experiments from germplasm banks as they store pure forms of plant materials (without any impurities and pathogen-free plant samples) so that we get good results and we are not performing any experiments on locally grown plant variety.