In vitro methods of germplasm conservation

In vitro conservation of plant germplasm

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.

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.

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.

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.

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.

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 CO₂is 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.