Methods of Preservation of Germplasm

Read this article to learn about the various methods of preservation of germplasm.

In the recent years, with the tremendous increase in the population, pressure on the forest and land resources have increased which has resulted in the decline in the population of medicinal and economically important plant species.

Even some of the plant species are at the verge of vanishing because of severe threat to their natural habitat due to human interference. Such species are termed as ‘threatened’ species.

Thus, attempt has been made in the recent years at the national and international levels to protect and preserve plant species, threatened plants as well to those plant species which are not in use today but may serve as important resource for future breeding programme.

In India, Botanical Survey of India has released three volumes (Red Data Book) enlisting the threatened plants of Indian subcontinent. There are several other organisations, which are engaged in the protection and preservation of such plants. Some of these organisations are Indian Council of Agricultural Research, New Delhi; International Bureau of Plant Genetic Resources, Nottingham, U.K.; National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi; Council of Scientific and Industrial Research, New Delhi and Botany Departments of various Universities.

Attempts have been made to conserve the germplasm by preserving the genetic material, which can be done by two ways:

A. In situ preservation

B. Ex situ preservation

A. In situ Preservation:

It aims at the preservation of the germplasm in their natural environment by establishing bio-sphere reserves, national parks, gene sanctuaries etc.

Limitations:

The limitation of this type of preservation is the risk of declination of the preserved species due to environmental hazards.

B. Ex situ Preservation:

This is the chief mode of preservation of germplasm. Providing the suitable condition in the gene bank preserves the genetic materials in the form of seed or in vitro cultures. But for being successful in establishment of gene bank considerable knowledge of genetic structure as well as elements influencing them are necessary.

Usually seeds form the most common material to conserve plant germplasm in the seed propagated plants. But this method has certain limitations such as:

Loss of seed viability with passage of time.

Seed destruction due to seed borne pathogens, pest etc.

This method is confined only to seed propagating plants.

In contrast to seed propagating plants, the vegetatively propagated plants are preserved in vitro as shoots, meristems, embryos etc. The advantages of in-vitro preservation over in situ preservation are –

Large amount of material can be preserved in small area.

It overcomes the destruction due to environmental hazards.

It provides large amounts of plant material for culturing.

Using either of the following techniques, preservation of germplasm can be achieved.

Gene Bank:

Germplasm or genetic resources are stored in the form of ‘seeds’ in a specialized place known as ‘gene bank’. Thus, a gene bank contains seeds (and not DNA/genes) of a plant species for future use. Collection of genetic resources at one place for use and distribution to others has a long history. By the end of nineteenth century, the US department of Agriculture had setup a plant exploration section. This started collection and import of new germplasm to the US. This leads to cultivation of soybean in the US, which has become a multi-million dollar soybean oil and protein industry.

As concern about the conservation of genetic resources increased, the collection and storage of material for immediate use was changed to storage for future use. In addition, the aim was not only to collect popular cultivated varieties, but also the land races and wild relatives of crop plants. This idea was initiated by N.I. Vavilov, who set up a large gene bank in Leningrad. By 1980s there were dozens of gene bank in seventy countries around the world.

Some of these collections were started at international research institutes like IRRI, Phillipines, CIMMYT, Mexico, ICRISAT, India, IBPGR, Rome and IARI, India. All are under the general co-ordination of the international board for plant genetic resources (IBPGR) at Food and Agriculture Organization, Rome.

All materials sent to gene bank are given an identification number called an accession number. The bank grown all seeds and compares their characters. This is a big task to manage seeds, data and information and computers are used for these purposes.

New germplasm of existing cultivated plants was collected by IBPGR, from Africa and west of Asia, e.g., perennial chick pea from Morocco, perennial oat from Algeria, and wild sorghum from Namibia.

For most important crops, the plant part that is stored in a gene bank is the seed. In vegetatively propagated crops, the seeds are either not produced or are not suitable. Therefore in such plants cuttings (many fruit trees) or tissue cultures cells/tissues are stored. Clonal propagation of stored tissue culture cells is the way, forest genetic resources are stored.

Seeds with the high oil content or large size are usually stored in bags at -18 °C. These seeds must be tested for germination every 10 years. Small seeds with less oil (e.g., cereal grains) are stored in liquid nitrogen at -196°C. Storage at such a low temperature reduces the cost of germination work; as such seeds may be stored for decades without germination testing.

Cryopreservation:

Cryopreservation [preservation in the frozen state (cryo means extreme cold, derived from Latin word kruos = ‘frost’)] is based on the reduction and subsequent arrest of metabolic functions of biological material by imposition of ultra-low temperature.

At the temperature of liquid nitrogen (-196°C) almost all the metabolic activities of cells are ceased and the sample can then be preserved in such state for extended periods. However, only few biological materials, in their natural state, can be frozen to sub-freezing temperatures without adversely affecting the cell viability. Knowledge about the chemicals having cryopreservative properties such as glycerol and dimethyl sulfoxide facilitated the development of effective cryopreservation technique.

Need for Cryopreservation:

The main application and objective of developing cryopreservation technology is preservation of valuable genetic resources, especially of vegetatively propagated and also of those species which have short lived seeds. Therefore, it is imperative that use of cryopreservation technology requires efficient regeneration protocols through tissue culture of the species. Cryopreservation of endangered species is one of the most important objectives of NBPGR, New Delhi.

Endangered species for which micro-propagation techniques have been developed in India are listed in Table 27.6. The regeneration methods using apical meristems are advantageous because these are simple and there are no chances of genetic variation. It is essential that tissue culture methods do not create genetic variability.

In contrast to above, the cases where genetic variability has been induced in the cultures, this variability need preservation for use in the future. Therefore, cryopreservation technique is equally good for preserving the genetic resources of existing genotypes and also of new variants.

Procedure of Cryoprotection and Pretreatment:

Some form of cryoprotection is necessary for cryopreservation of plant material unless they are naturally dehydrated, as in the case of dormant vegetative buds in the winter, or artificially cold acclimated. Several chemicals such as dimethyl sulphoxide (DMSO), glycerol, various sugars and sugar alcohols protect living cells against damage during freezing and thawing (means to become unfrozen or warm after preservation at ultra low temperatures). These compounds lower the temperature at which freezing first occurs and can alter the crystal habit of ice when it separates.

The colligative properties of the cryoprotectants minimize the harmful action of electrolyte concentration resulting from conversion of water into ice. High solubility in aqueous phase and low toxicity to the cells are the two essential characteristics for cryoprotectants, e.g., DMSO, methanol, glycerol and sugars, sugar alcohols, high molecular weight polymers as dextran, polyvinyl pyrrolidone, hydroxy ethyl starch.

The cells require different pretreatment periods with different compounds for proper cryoprotection. DMSO enters more rapidly than glycerol and therefore requires shorter period for treatment. Most of the cryoprotectants exhibit varying degree of cytotoxicity at higher concentrations. Generally, DMSO at 5 to 10% and glycerol at 10 to 20% are used as cryoprotectants. Sometimes, a mixture of cryoprotectants can also be used to improve the efficacy.

Addition of osmotically active compounds in the culture medium such as mannitol, sorbitol, sucrose and proline increase the freezing resistance of the cells. These compounds mainly act by their dehydration effect Sorbitol has been successfully used as an osmotic agent as well as cryoprotectant for Glycine max, Datura innoxia, Brassica napus, and Daucus carota.

Freezing Methods:

Methods used by different workers for cryopreservation of various plant materials can be categorized as:

(i) Slow freezing,

(ii) Rapid freezing and

(iii) Droplet-freezing.

i. Slow Freezing:

Methods of slowly freezing biological specimens are based on the physicochemical events occurring during the freezing. When plant cells are cooled progressively, ice crystal formation is usually initiated extra-cellularly. It is presumed that plasma membrane acts as a barrier that prevents the ice crystal formation in the cytoplasm.

In absence of ice crystals, cytoplasm remains super cooled. On further lowering of temperature, the concentration of extra­cellular liquid increases as more water is converted to ice.

Since the vapour pressure of the frozen solution is lower than the same concentration of the super cooled liquid, the vapour pressure of slowly cooled cells reach equilibrium with external ice by efflux of water. Thus, slow freezing prevents the intracellular ice formation and consequently freezing injury is prevented.

It is believed that slow freezing increase the concentration of cytoplasm and increased dehydration increases the survival of cells. There are different methods of obtaining protective dehydration, including slow cooling at constant or varying cooling rates, or keeping the samples at one or more intermediate subzero temperatures.

The development of efficient slow freezing method depends upon several factors like cooling rates, pretreatment and cryoprotection, type and physiological state of the material, and the temperature prior to immersion in liquid nitrogen.

The most commonly used methods for the cryopreservation of plant cells generally involves regulated slow cooling at a constant rate of 0.5 to 2 °C/min. to terminal temperature between -30 °C to -40 °C followed by storage in liquid nitrogen (Table 27.7).

ii. Rapid Freezing:

Rapid freezing is unsuitable for the cryopreservation of cell cultures, it is employed to cryopreserve shoot tips of carnation, potato, strawberry and several others. Rapid freezing is accomplished by direct immersion of the cryoprotectant-treated specimens in liquid nitrogen. The cooling rate in this method is very high, usually several hundred degrees per minute. At such high cooling rates, the intracellular fluids do not have sufficient time to equilibrate with the external ice with the possibility of intracellular ice formation, which is considered to be lethal for cells and somatic embryos of oil palms.

iii. Droplet Freezing:

In this method the cryoprotactant treated meristems are dispensed in droplets of 2-3 µl on an aluminum foil in a petriplate. The specimens are frozen by slow cooling (0.5°C/min.) to a subzero temperature between -20 to -40 °C prior to immersion in liquid nitrogen.

iv. Storage, Thawing and Re-growth:

Material can be kept stored in liquid nitrogen (-196 °C) or in its vapour (-150 °C). Rapid thawing is recommended for most cryopreservation methods. The basis of applying rapid thawing is to avoid the damaging ice re-crystallization which may occur during slow warming. In general thawing is carried out by removing the sample by liquid nitrogen storage and transferring in a water bath (34-40 °C) for about 1-2 min or until material is warmed up.

Re-growth of cryopreserved specimens is the most reliable and accurate estimate of viability. The other technique used for vitality test of the cells such as fluorescein diacetate stain, triphenyl tetrazolium chloride test etc. may provide a quicker method of testing cell viability. The further growth and regeneration of the cryo-preserved cells after thawing will be like normal cells. However, care should be taken in handling, plating and subculture of such cells.

v. Freezing Apparatus:

Various types of cryostate and freezing units are available by which different rates of cooling can be easily regulated (Fig. 27.2).

The culture subjected to ultra cooling can be stored at -196 °C in the liquid nitrogen container for various lengths of time and can be taken out and thawed when required.

vi. Applications of Cryopreservation:

1. Conservation of genetic uniformity.

2. Preservation of rare genomes.

3. Freeze storage of cell cultures and cell lines.

4. Maintenance of disease free material.

5. Cold acclimation and frost resistance.

6. Retention of morphogenetic potential in long-term cultures.

7. Slow metabolism and aging.

In spite of the information and technology available for in vitro storage of several crop species, in vitro active gene banks exist so far only for potato at International Potato Cenre, Lima, Peru and casava at Centro International de Agricultura Tropical (CIAT), Cali, Colombia. Recently, efforts have been made under the auspices of International Board for Plant Genetic Resources (IBPGR) to exploit the full potential of in vitro storage methods employing meristem and shoot tip cultures.

The initiation of a pilot project by collaborative efforts of IBPGR and CIAT, using cassava as a model system, for assessing the potential and feasibility of establishing and running an in vitro active gene bank is a first significant step. Under the project more than 4000 clones of cassava are being maintained in vitro under minimal growth conditions. In India, storage facility has been created at National Bureau of Plant Genetic Resources (NBPGR), New Delhi for germplasm preservation using in vitro methods.