In Vitro Methods of Germplasm Conservation

In in vitro methods of germplasm conservation, the first germplasm conservation method is the Cryopreservation. Rest of the methods are as follow:

In Vitro Methods of Germplasm Conservation

(a) Cryopreservation in Cryopreservation (Greek- krayos-frost)

In this case the cells are preserved in the frozen state. The germplasm is stored at a very low temperature using

Solid carbon dioxide (at -790C)
Using low temperature deep freezers (at -800C)
Using vapour nitrogen (at -1500C)
Liquid nitrogen (at-1960)

The cells stay in completely inactive state. Thus, they can be conserved for long periods. Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide etc. Are added during the cryopreservation. These are called cryoprotectants and prevent the damage caused to cells (by freezing or thawing) by reducing the freezing point and super cooling point of water. This is the first  in vitro method of germplasm conservation.

(b) Cold Storage

The second in vitro method of germplasm conservation is cold storage. It is a slow growth germplasm conservation method. It conserves the germplasm at a low and non-freezing temperature (1 to 9°C). The growth of the plant material is slowed down in cold storage in contrast to complete stoppage in cryopreservation. Thus, it prevents cryogenic injuries. Long term cold storage is simple, cost effective. It yields germplasm with good survival rate. Virus free strawberry plants could be preserved at 10°C for about 6 years. several grape plants have been stored for over 15 years by using a cold storage at temperature around 9°C and transferring them in the fresh medium every year.

(c) Low Pressure and Low Oxygen Storage

The third in vitro method of germplasm conservation is low- pressure storage, the atmospheric pressure surrounding the plant material is reduced. In the low oxygen storage, the oxygen concentration is reduced. The lowered partial pressure reduces the in vitro growth of plants. In the low-oxygen storage, the oxygen concentration is reduced. The lowered partial pressure of oxygen below 50 mmHg reduces plant tissue growth. Due to the reduced availability of O₂, and reduced production of CO₂, the photosynthetic activity is reduced. It inhibits the plant tissue growth and dimension. This method has also helped in increasing the shelf life of many fruits, vegetables and flowers.

In Vitro Methods of Germplasm Conservation Limitations

The in vitro methods of germplasm conservation has several limitations such as short-lived seeds, seed dormancy, seed- borne diseases, and high inputs of cost and labour. The techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storages help us to overcome these problems.

Advantages and Limitations of Germplasm Storage

The in vitro methods of germplasm conservation storage have great advantage for clonally propagated crops and for crops having recalcitrant/short-lived seems. The potential advantages of these techniques are as follows:

(i) Requirement of relatively very small space,

(ii) Storage of germplasm free from diseases, insects and other pathogens, and weeds,

(iii) Storage over long periods,

(iv) Reduced risk of errors in labeling, etc. In addition, such materials are ‘clean’ sources of ‘nucleus seed’,

(v) They are ideal for germplasm exchange.

However, these approaches suffer from the following disadvantages:

(i) Sophisticated facilities are required (particularly for freeze preservation and DNA cloning),

(ii) They demand a greater skill in handling and maintenance than the conventional techniques,

(iii) Even shoot tip derived plants may show genetic instability, at least in some plant species.

(iv) Cells/tissues become damaged during cryopreservation.

(v) Even DNA may become damaged due to cryopreservation under suboptimal conditions.

(d) Slow Growth Cultures

Slow-growth of plantlets ill vitro provides an attractive alternative to freeze preservation of germplasm as it is simpler, cheaper and very effective. Slow growth may be achieved by maintaining the plantlets either at a low temperature (4-9°C or Ca. 15°C) or on a medium having high osmotic concentration (e.g., 20% sorbitol or sucrose) or both.

In addition, the nutritional status of the medium may be lowered to restrict the growth of plantlets. Under the conditions of slow-growth, cultures may be attended to only once in several months. Its subculture may, be necessary only after long periods, once every 12-36 months.

For example, grape plantlets stored at 9°C need to be transferred after 12 months; they have been thus maintained for more than 15 years. It has been estimated that 6 replicates of 800 grape varieties can be maintained in only 2 m2 laboratory space, while it would require in the field.

The slow-growth approach is being utilized for germplasm conservation of specified root, tuber and tree species by the NBPGR, New Delhi. A National Facility for Plant Tissue Culture Repository has been created for this purpose. It has so far developed the slow-growth protocols for ginger, garlic, banana, sweet potato, etc.

(e) Desiccated Somatic Embryos (SE) and Artificial Seeds

The techniques for desiccation of SE s and for production of desiccated, artificial seeds are now becoming available. The desiccated SE s and artificial seeds can be stored at low (4°C) or ultralow (-20°C) temperatures for prolonged periods in a manner similar to zygotic seeds.

This approach is yet to be evaluated for such an application. It involves complicated desiccation procedures. It can be applied only to those species/genotypes where SE regeneration occurs in adequate frequency.

(f) DNA Clones

Germplasm can also be conserved in form of DNA segments cloned in a suitable vector, e.g., cosmids, phasmids or YACs. The technique is highly sophisticated, technically demanding and expensive.

It is likely to be used for conservation of valuable genes or DNA segments from threatened species. It can also be used for the conservation of the entire genomes of various germplasm lines of different species.

Comparison of Three Approaches for in Vitro Germplasm Conservation

Feature	Cryopreservation	Slow-Growth	DNA Clones
Tissue/Organ conserved	Shoot-tips, zygotic or somatic embryos, cells, protoplasts	Slow growing roots	DNA pieces as phage clones
Metabolic activity	Nil	Slow	Nil
Storage temperature	-196°C	4-9 or ~15°	4°C; in lycophylised state
Storage in	Liquid nitrogen refrigerators	Ordinary refrigerators	Ordinary refrigerator/deep freeze
Attention needed during storage	Replenishing liquid nitrogen	Subculture every 6-36 months	Virtually nil