**3.5.2 Vitrification**

A simplified methodology for vitrification is given below (Yamuna 2007).

1. Shoots (1-2mm)/ somatic embryos were excised and cultivated on MS medium supplemented with 0.3 M sucrose for 24h at 250C.

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

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,

The *in vitro* plants already established were used as mother plants for source of explants. This in turn facilitates the reduction in size of the plantlets and smaller somatic embryos

1. Suspend *in vitro* grown shoots/ somatic embryos in MS basal medium supplemented

2. Drop the mixturecontining microshoots, with a sterile pipette into 0.1M CaCl2 solution containing 2M Glycerol and 0.4M sucrose and left for 20 min to form beads about 4 mm

3. Preculture the encapsulated shoots – stepwise - on MS medium enriched with different

4. Place the precultured beads on sterile fitter paper in Petridishes (diameter 90mm) and dehydrated by air drying on a flow bench (at room temperature and humidity) for

5. Measure the water content of the beads was by weighing them prior and after drying in

6. Transfer the dehydrated beads into a 2 ml cryovial (ten beads per tube) and directly

1. Shoots (1-2mm)/ somatic embryos were excised and cultivated on MS medium

concentration of 0.3, 0.5, 0.75 and 1.0M for four days with one day on each.

encapsulation- dehydration, encapsulation- vitrification and vitrification.

A simplified methodology for vitrification is given below (Yamuna 2007).

with 4% (w/v) Na alginate, 2M Glycerol and 0.4 M sucrose.

periods of 0-10 h to determine the optimal dehydration time.

A simplified methodology for vitrification is given below (Yamuna 2007).

supplemented with 0.3 M sucrose for 24h at 250C.

in diameter, each bead containing at least one shoot.

research laboratories

**3.5.1 Encapsulation - Dehydration** 

an oven at 800C for 48h.

**3.5.2 Vitrification** 

immerse in liquid nitrogen for 24h.

which made them suitable for cryopreservation.


#### **3.5.3 Encapsulation – Vitrification**

A simplified methodology for encapsulation - vitrification is given below (Yamuna 2007).


#### **3.6 Thawing and recovery of conserved materials**

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

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 for multiplication.

#### **3.7 Genetic stability of conserved materials**

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 the genetic stability of *in vitro* materials.

Cryopreservation of Spices Genetic Resources 465

**Application Technique Reference** 

cryopreservation Plantlets and shoot tips

Disease eradication Shoot cultures, shoot

cryopreservation Plantlets and shoot tips

cryopreservation Plantlets and shoot tips

Propagation Somatic embryo

**Cardamom** *(Elettaria* 

*Zingibe***r spp.** 

*Curcuma* **spp** 

*Vanilla* **spp.** 

**Herbal spices** 

*Capsicum* 

*cardamomum* **Maton***)*

Slow growth storage and

Slow growth storage and

Slow growth storage and

Slow growth storage and

**Fennel** *(Foeniculum vulgare)*

microbullbets Cryopreservation Shoot tips Niwata 1995

regeneration

Cryopreservation Synseeds Sharma *et al*., 1994

cryopreservation Plantlets and shoot tips Ravindran *et al*., 2004 ;

Cryo preservation Synthetic seeds Ravindran *et al.,* 2004 Pollen Cryo preservation Pollen Minoo, 2002; Minoo *et al*

Slow growth storage *In vitro* plantlets Nirmal Babu *et al*.1996

Cryopreservation Pollen Alexander *et al.,* 1991

Cold storage Embryogenic Umetsu *et al.,* 1995

Cryopreservation Seed Peter *et al* 2002 ; Ravindran

Cryopreservation Pollen Rajasekharan and Ganeshan,

Disease eradication Apical meristem Cereveta and Madrigal, 1981

Disease eradication Meristem culture Nadagauda *et al.* 1983 Cryopreservation Seeds Chaudhury and Chandel,

1995

buds Balachandran *et al* .,1990

2007

2011

*et al* 2004

2003

Ravindran *et al*., 2004; Nirmal Babu *et al*., 2007;

Hosoki and Sagawa ,1977,

Nirmal Babu, 1997

Ravindran *et al*., 2004; Nirmal Babu *et al*., 2007; Yamuna *et al* 2007 ; Yamuna

Nirmal Babu *et al*., 2007

Ravindran *et al*., 2004; Nirmal Babu *et al*., 2007, Minoo and Babu 2009

Yamuna 2007

RAPD, ISSR and SSR analysis can be done to evaluate genetic fidelity of the cryopreserved lines of Spices. DNA isolation can be done as per CTAB method (Ausubel *et al*., 1995 or Sambrook *et al*. 1989). RAPD and ISSR, SSR profiles were developed as per the method suggested by Williams *et al*., (1990), Nirmal babu *et al*., (2003, 2007) and Ravindran *et al*., (2004).

Morphological characters coupled with RAPD profiles using 24 operon primers have indicated genetic fidelity among randomly selected micropropagated plants of Subhakara and Aimpiriyan, indicating that micropropagation protocol can be used for commercial cloning of black pepper (Nirmal Babu *et al.,* 2003). Genetic uniformity of micropropagated *Piper longum*  using RAPD profiling was reported by Ajith (1997) and Parani *et al.* (1997) for conservation.

Peter *et al* (2001) and Ravindran *et al* (2004) reported that the conserved materials of all the species conserved by them showed normal rate of multiplication when transferred to multiplication medium after storage. The normal sized plantlets when transferred to soil established with over 80% success. They developed into normal plants without any deformities and were morphologically similar to mother plants. RAPD profiling of these conserved plants also showed their genetic uniformity.

Ravindran *et al* (2004), Yamuna *et al* (2007) and Yamuna (2007) reported genetic uniformity was observed in cryo preserved and recovered plants of cardamom, ginger, black pepper and endangered species of Piper, *P. barberi* based on RAPD and ISSR profiling.
