**3.5 Cryo preservation**

For long-term conservation of the problem species, cryopreservation is the only method currently available. Dramatic progress has been made in recent years in the development of new cryopreservation techniques and cryopreservation protocols have been established for over 100 different plant species.

Cryopreservation is an attractive option for long-term storage. Liquid nitrogen (–196°C) is routinely used for cryogenic storage, since it is relatively cheap and safe, requires little maintenance and is widely available. Below –120°C the rate of chemical or biophysical reactions is too slow to cause biological deterioration (Kartha 1985). Only in the long term might there be a small risk of ionising radiation causing genetic changes in materials stored at cryogenic temperatures (Grout 1995).

An array of plant material could be considered for cryopreservation as dictated by the actual needs *vis-a-vis* preservation. These include meristems, cell, callus and protoplast cultures, somatic and zygotic embryos, anthers, pollen or microspores and whole seeds (Withers, 1985; Kartha, 1985).

Plant germplasm stored in liquid nitrogen (-196°C) does not undergo cellular divisions. In addition, metabolic and most physical processes are stopped at this temperature. As such, plants can be stored for very long time periods and both the problem of genetic instability and the risk of loosing accessions due to contamination or human error during subculturing are overcome. Most cryopreservation endeavours deal with recalcitrant seeds, *in vitro* tissues from vegetatively propagated crops, species with a particular gene combination (elite genotypes) and dedifferentiated plant cell cultures. Care must be taken to avoid ice crystallisation during the freezing process, which otherwise would cause physical damage

Cryopreservation of Spices Genetic Resources 463

2. The treated explants were then cultured on MS medium supplemented with sucrose at

3. After pretreatments explants were transferred to a cryovial with 1.8 ml of loading

4. Different incubation periods in PVS2 (40-100 minutes) were tested for osmoprotected

5. Cryovials containing 8-10 explants were directly immersed in liquid nitrogen and kept

A simplified methodology for encapsulation - vitrification is given below (Yamuna 2007). 1. Suspend pre-cultured shoots (1-2mm)/ somatic embryos with 2-3 apical domes on 0.3M sucrose for 16h in MS basal medium supplemented with 4% sodium alginate and 0.3 M

2. Dispense the mixture including shoots, were with a sterile pipette into MS medium supplemented with 0.1M CaCl2 and 0.4 to 1.0M sucrose, with or without 2M Glycerol

3. The encapsulated and osmo-protected shoots were dehydrated with 20 ml PVS2 in a 100 ml Erlenmeyer flask at 250C and plunged into LN and held for at least 24 h at -

After LN storage, cryovials warm rapidly in a 40 0C water bath for 2-3 minutes. The solution was drained from the cryovials and replace twice at 10 min intervals with 1 ml 1.2 M sucrose solution in the case of encapsulation- vitrification and vitrification methods. The composition of recovery medium was MS/WPM/SH basal medium supplemented with

In the Encapsulation - dehydration, Encapsulation - vitification and vitrification procedures, surviving shoots can be identified by greening of explants following 2 weeks of post culture. Regrowth can be defined as the shoots that regenerated to shoots in 6 weeks of postculture. Elongated shoots can be used for micropropagation and rooting and subculture was done every 4 weeks. For rooting well grown shoots can be transferred to solid MS medium used

An important prerequisite for any conservation technique is that the regenerants produced from the conserved material should be true-to-type. There are ample evidences to indicate that under certain culture conditions the materials undergo genetic changes (somaclonal variations) and as a consequence lose their integrity and uniformity. This would be highly undesirable in spices varieties where the purpose is not only to conserve a genotype but also retain its specific quality traits. Thus testing for the genetic stability of *in vit*r*o* conserved materials is of utmost importance. Besides morphology, cytology and isozyme profiling sophisticated biochemical and DNA-based techniques have enabled more critical analysis of

0.75 M for 1 day in the same conditions.

explants

for 24 h.

sucrose.

1960C.

for multiplication.

**3.5.3 Encapsulation – Vitrification** 

solution (2 M Glycerol + 0.4 M sucrose) and kept for 15 min.

gently shaken (20 rpm) on a rotary shaker for 1h at 250C.

**3.6 Thawing and recovery of conserved materials** 

2.22 – 4.44 μM and BA, 2.69- 5.37 μM NAA.

**3.7 Genetic stability of conserved materials** 

the genetic stability of *in vitro* materials.

to the tissues. The existing cryogenic strategies rely on air-drying, freeze dehydration, osmotic dehydration, addition of penetrating cryoprotective substances and adaptive metabolism (hardening), encapsulation, vitification or combinations of these processes.

Cryopreservation methods have been developed for more than 80 different plant species in various forms like cell suspensions, calluses, apices, somatic and zygotic embryos (Kartha and Engelmann, 1994; Engelmann, 1997, 2000, Engelmann *et al* 1994, 1995). However, their routine utilisation is still restricted almost exclusively to the conservation of cell lines in research laboratories

For small volumes, long-term storage is practicable through storage of cultures in cryopreservation at ultra-low temperature, usually by using liquid nitrogen (-196oC). At this temperature all cellular divisions and metabolic processes are virtually halted and consequently, plant material can be indefinitely stored without alteration or modification.

The normal approach of tissue culture is to find a medium and set of conditions that favour the most rapid rate of growth with a subculture interval of 20 – 30 days. For cryopreservation storage biological materials are stored in liquid nitrogen for long term with out subculturing. Cryopreservation, i.e., the storage of biological material at ultra low temperature usually that of liquid nitrogen (-1960C) can be achieved by different techniques like direct freezing, encapsulation- dehydration, encapsulation- vitrification and vitrification.
