**4. Status of cryo conservation in spices**

Reports on cryopreservation of spices are meager and limited. The present status of cryo preservation in major spices is given Table 2. The number of accessions conserved in cryo genebank at the National Bureau of Plant Genetic Resources (NBPGR), New Delhi are given in Table 3.


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*.,

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

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

Reports on cryopreservation of spices are meager and limited. The present status of cryo preservation in major spices is given Table 2. The number of accessions conserved in cryo genebank at the National Bureau of Plant Genetic Resources (NBPGR), New Delhi are given

propagation Shoot Culture Leaf/root Broome and Zimmerman,

Cryopreservation Seeds Chaudhury and Chandel

Cryopreservation Seed Decruse and Seeni, 2003

1978

,1994

themotherapy Conci and Nome, 1991

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

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

Yamuna 2007

Yamuna 2007

and endangered species of Piper, *P. barberi* based on RAPD and ISSR profiling.

**Application Technique Reference** 

**and related species** Meristem culture Philip *et al.,*<sup>1992</sup>

conserved plants also showed their genetic uniformity.

**4. Status of cryo conservation in spices** 

Cryopreservation synseeds

cryopreservation Plantlets and shoot tips

Disease eradication Meristem culture and

Cryopreservation Shoot culture, Keller 1991

**Black pepper (***Piper nigrum* **)**

Disease eradication

Slow growth storage and

(2004).

in Table 3.

*Allium* **Spp** 


Cryopreservation of Spices Genetic Resources 467

Fig. 1. Cryopreservation of black pepper somatic embryos by encapsulation dehydration. a) Somatic embryos used for cryopreservation, b) Somatic embryos encapsulated in Naalginate, c) Encapsulated and dehydrated somatic embryos, d) Viable somatic embryo stained in red colour after cryopreservation, e), f), g), h) & i) Various stages of development of somatic embryos to plantlet after cryopreservation, j) Fully developed plantlet from a somatic embryo cryopreserved by encapsulation dehydration, k) A cluster of somatic embryos at different stages of development, originated from an embryogenic line after

Yamuna (2007) reported the effect of encapsulation-dehydration and vitrification methods on survival of cryo preserved somatic embryos in black pepper. In encapsulation dehydration treatment, the best survival rates (62 %) of somatic embryos was obtained after freezing, by preculturing in 0.7 M sucrose (direct) for 1 day, followed by dehydration in the

cryopreservation


\*Ashmore, 1997, 2002 and Nirmal Babu *et al*, 1999, 2007, Yamuna 2007; Yamuna et al 2007

Table 2. Present status of information on cryo conservation of spices


Source: Annual Report NBPGR 2010-11

Table 3. Present status of Spices in *in vitro* and Cryo genebank at NBPGR

#### **4.1 Black pepper and related species**

Cryopreservation of black pepper (*Piper nigrum* L.)seeds in liquid nitrogen (LN2) was reported by Choudhary and Chandel, (1994), and Choudhury and Malik (2004). Pepper seeds are recalcitrant and the seed viability decreases with reduction in moisture content. Seeds desiccated to 12% & 6%moisture contents were successfully cryopreserved in liquid nitrogen at –1960C, with a survival rate of 45% & 10.5% respectively (Chaudhury and Chandel 1994).

suspension cells

Cryopreservation somatic embryos Elena *et al.,* (2010)

Cryopreservation Somatic embryos Leigh and Remi 2003

Slow growth Encapsulated beads Mandal *et al* (2000)

Cryopreservation Hairy root cultures Phunchindawan *et al*

**Species No.of accessions** 

\*Ashmore, 1997, 2002 and Nirmal Babu *et al*, 1999, 2007, Yamuna 2007; Yamuna et al 2007

Table 2. Present status of information on cryo conservation of spices

Spices and industrial crops 380 accessions

Medicinal and Aromatic plants 169 accessions

Spices and Condiments 148 accessions Medicinal and Aromatic plants 5 accessions

Table 3. Present status of Spices in *in vitro* and Cryo genebank at NBPGR

Cryopreservation of black pepper (*Piper nigrum* L.)seeds in liquid nitrogen (LN2) was reported by Choudhary and Chandel, (1994), and Choudhury and Malik (2004). Pepper seeds are recalcitrant and the seed viability decreases with reduction in moisture content. Seeds desiccated to 12% & 6%moisture contents were successfully cryopreserved in liquid nitrogen at –1960C, with a survival rate of 45% & 10.5% respectively (Chaudhury and

Total 702

Shoot tips Shatnawi *et al* (2004).

Encapsulated calluses Chand *et al* (2000);

( 7 genera, 27 species)

( 21 genera, 28 species)

Baghdadi et al., (2010)

**Application Technique Reference** 

**Coriander** 

*Ocimum* **spp** 

*Crocus* **spp.**

**(***Coriandrum sativum***)** 

**Mint** *(Mentha* **spp.)** 

*Syzygium francissi*

*Armoracia rusticana*

Maintained as in vitro cultures

Maintained in cryo bank

Chandel 1994).

Source: Annual Report NBPGR 2010-11

**4.1 Black pepper and related species** 

Fig. 1. Cryopreservation of black pepper somatic embryos by encapsulation dehydration. a) Somatic embryos used for cryopreservation, b) Somatic embryos encapsulated in Naalginate, c) Encapsulated and dehydrated somatic embryos, d) Viable somatic embryo stained in red colour after cryopreservation, e), f), g), h) & i) Various stages of development of somatic embryos to plantlet after cryopreservation, j) Fully developed plantlet from a somatic embryo cryopreserved by encapsulation dehydration, k) A cluster of somatic embryos at different stages of development, originated from an embryogenic line after cryopreservation

Yamuna (2007) reported the effect of encapsulation-dehydration and vitrification methods on survival of cryo preserved somatic embryos in black pepper. In encapsulation dehydration treatment, the best survival rates (62 %) of somatic embryos was obtained after freezing, by preculturing in 0.7 M sucrose (direct) for 1 day, followed by dehydration in the

Cryopreservation of Spices Genetic Resources 469

Fig. 2. Cryopreservation of *Piper barberi* by encapsulation vitrification. a) *In vitro* culture of *P. barberi*, b) & c) Shoot tips encapsulated in Na-alginate, arrow indicates shoot tip used as explants, d), e), f) & g) Various stages of development of cryopreserved shoot tips after post

culturing, h) Regenerated plantlets after 3 months of post culturing

laminar air flow for 6 h which resulted in 21 % moisture content. In the vitrification procedure, the somatic embryos were precultured for 3 days on SH basal medium containing 0.3 M sucrose and subjected to vitrification treatment for 60 minutes at 250C resulted in 71 % survival after cryopreservation. The study concluded that the embryogenic lines of *Piper nigrum* cultivar karimunda can be successfully cyopreserved following an encapsulation dehydration/desiccation procedure (62 % success). This success rate can be enhanced to 71 % using a vitrification/one step freezing in liquid nitrogen (Fig. 1).This was mainly because of the nature of somatic embryos which is more suitable to cryopreservation compared to shoot buds. The genetic stability of the conserved somatic embryos was proved by RAPD and ISSR profiling. Cryopreservation of encapsulated shoot buds of endangered *Piper barberi* was reported by Peter *et al* (2001) and Ravindran *et al* (2004).

Encapsulated shoot tips of *Piper barberi* were cryopreserved with 60% success using vitrification technique. In encapsulation vitrification the encapsulated shoot tips were precultured on MS medium, supplemented with 0.3 M, 0.5 M and 0.7 M sucrose (pH 5.8) for three days followed by dehydration with PVS2 solution (100%) at 00 C for 3 hours. After dehydration the beads (10 encapsulated shoot tips in 0.8 ml PVS2 solution per 1.5 ml cryotube) were frozen rapidly by direct immersion in to liquid nitrogen (- 196 0C) and kept for one hour (Peter *et al* 2001 and Ravindran *et al* 2004). Yamuna 2007 also reported that studies on cryopreservation of endangered *P.barberi* shoot tips revealed that, the encapsulation- vitrification procedure produced higher survival (70 %) of cryopreserved shoot tips (Fig. 2) compared to encapsulation - dehydration which gave 40 % survival. Genetic fidelity studies showed that the regenerated plants were similar to the controls. Thus encapsulation - vitrification as a simple and efficient method for long term preservation of *P.barberi* propagules.
