**4.6 Seed herbal and other spices**

Elena *et al.,* (2010) successfully cryopreserved coriander (*Coriandrum sativum* L.) somatic embryos using sucrose pre-culture and air desiccation procedure utilized embryo clumps (ECs). The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration. Plants could be regenerated from cryopreserved seeds of Anise.( Peter *et al* 2001).

Successful Cryopreservation of seeds, meristems, somatic or zygotic embryos were reported in *Allium* Spp (Niwata, 1995, Hyung *et al* 2003, Haeng *et a*l 2003, 2004.2005, Jung *et al* 2005, Gayle *et al* 2004). Preliminary success was reported in cryo preservation of Mint (Leigh and Remi 2003).

Most of the reports are confined to a few genotypes and hence the techniques standardized needs to be extended to more genotypes before adopting them for routine conservation. Reports of cryoconservation of spices like *Ocimum, Lavendula, Salvia* are available from National Bureau of Plant Genetic Resources (NBPGR), New Delhi.

Plants could be successfully regenerated (Fig 8) from cryopreserved seeds of capsicum (Peter *et al* 2001 and Ravindran *et al* 2004). Alexander *et al* (1991) and Rajasekharan and Ganeshan. (2003) reported freeze preservation of capsicum pollen (*Capsicum annuum*) in

Elena *et al.,* (2010) successfully cryopreserved coriander (*Coriandrum sativum* L.) somatic embryos using sucrose pre-culture and air desiccation procedure utilized embryo clumps (ECs). The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration. Plants could be regenerated from cryopreserved seeds of

Successful Cryopreservation of seeds, meristems, somatic or zygotic embryos were reported in *Allium* Spp (Niwata, 1995, Hyung *et al* 2003, Haeng *et a*l 2003, 2004.2005, Jung *et al* 2005, Gayle *et al* 2004). Preliminary success was reported in cryo preservation of Mint (Leigh and

Most of the reports are confined to a few genotypes and hence the techniques standardized needs to be extended to more genotypes before adopting them for routine conservation. Reports of cryoconservation of spices like *Ocimum, Lavendula, Salvia* are available from

**4.5 Capsicum** 

liquid nitrogen (–1960C) for 42 months.

**4.6 Seed herbal and other spices** 

Anise.( Peter *et al* 2001).

Remi 2003).

Fig. 8. Successful germination of cryopreserved seeds of capsicum

National Bureau of Plant Genetic Resources (NBPGR), New Delhi.

Mandal *et al* (2000) reported propagation and conservation of four pharmaceutically important herbs, *Ocimum americanum* L. syn. *O. canum* Sims. (hoary basil); *O basilicum* L. (swett basil); *O. gratissimum* L. (shrubby basil); and *O. sanctum* L. (sacred basil) using synthetic seed technology. Synthetic seeds were produced by encapsulating axillary vegetative buds harvested from garden-grown plants of these four *Ocimum* species in calcium alginate gel. The gel contained Murashige and Skoog (MS) nutrients and 1.1-4.4 μ*M* benzyladenine (BA). Shoots emerged from the encapsulated buds on all six planting media tested. However, the highest frequency shoot emergence and maximum number of shoots per bud were recorded on media containing BA. Of the six planting media tested, both shoot and root emergence from the encapsulated buds in a single step was recorded on growth regulator-free MS medium as well as on vermi-compost moistened with halfstrength MS medium. Rooted shoots were retrieved from the encapsulated buds of *O. americanum, O. basilicum*, and *O. sanctum* on these two media, whereas shoots of *O. gratissimum* failed to root. The encapsulated buds could be stored for 60 d at 4°C. Plants retrieved from the encapsulated buds were hardened off and established in soil.

An efficient procedure for the *in vitro* propagation and cryogenic conservation of *Syzygium francissi* was developed by Shatnawi *et al* (2004). Shoot tips excised from *in vitro*-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 *M* sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for *in vitro* propagation and cryopreservation of shoot tips from *in vitro*-grown plantlets of *S. francissi* germplasm.

Hairy root cultures of *Armoracia rusticana* Gaertn. Mey. et Scherb. (horseradish) were successfully cryopreserved by two cryogenic procedures (Phunchindawan *et al.,* 1997). Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5*M* sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5*M* sucrose and 1*M* or 1.5*M* glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots

The effect of sucrose concentration and dehydration period on survival and regrowth of encapsulated calluses were also studied in 2 species of Crocus (Chand et al 2000). Highest survival (83.3; 88.9%) and regrowth (77.6; 83.3%) rates were obtained when encapsulated unfrozen calluses of *Crocus hyemalis* and *C. moabiticus* precultured with 0.1 M sucrose for two days without further air dehydration. After cryopreservation, the highest survival (55.6; 61.1%) and regrowth (16.7; 27.8%) rates were achieved when calluses of *C. hyemalis* and *C. moabiticus* were pretreated with 0.5 M sucrose for two days after two hours of dehydration. Viability of crocus decreased with increased sucrose concentration and dehydration period. Dehydration of encapsulated calluses of *C. hyemalis* and *C. moabiticus* with silica gel for one hour prior to freezing resulted in maximum rates of survival (77.8; 83.3%) and re-growth

Cryopreservation of Spices Genetic Resources 477

plant material, particularly for vegetatively propagated species. Cryopreservation is the only viable method available for long-term preservation of the both plant and animal origin species. As an ultimate aim of cryoconservation is the reintroduction of preserved material into the field, it is appropriate at this point to consider the concept of restoration a little more closely. In terms of ultimate ecosystem restoration, the possibilities raised by in vitro conservation, including cryoconservation, do not mean that species selection should merely take random advantage of what germplasm has or can be conserved as there are many genetic, physiological and phenotypic considerations to be taken into account (Kramer and

The establishment and maintenance of biological resource centers (BRCs) or germplasm conservatories requires careful attention to implementation of reliable preservation technologies and appropriate quality control to ensure that recovered cultures and other biological materials perform in the same way as the originally isolated culture or material. There are many types of BRC that vary both in the kinds of material they hold and in the purposes for which the materials are provided. All BRCs are expected to provide materials and information of an appropriate quality for their application and work to standards relevant to those applications. There are important industrial, biomedical, and conservation issues that can only be addressed through effective and efficient operation of BRCs in the long term. This requires a high degree of expertise in the maintenance and management of collections of biological materials at ultra-low temperatures, or as freeze dried material, to secure their long-term integrity and relevance for future research, development, and conservation. The application of cryogenic preservation in biotechnology and medicine has recently been a topic of interest. The use of cryogenic preservation in this area has given

Adams, R.P. and Adams, J.E., 1991. Conservation of Plant Genes: DNA Banking and In Vitro

Adams, R.P., 1988. The preservation of genomic DNA: DNA Bank Net. *Amer. J. Bot*., 75:

Adams, R.P., 1990. The preservation of Chihuahuan plant genomes through *in vitro* 

Adams, R.P., 1997.Conservation of DNA: DNA banking. In: *Biotechnology and Plant Genetic* 

Ajith, A., 1997. Micropropagation and genetic fidelity studies in *Piper longum* L. In:

biotechnology:DNA Bank*-*Net, a genetic insurance policy. In: *Third Symposium on Resources of the Chihuahuan Desert Region,* (Eds.) Powell, A.M., Hollander, R.R., Barlow, J.C., McGillivray, W.B. and Schmidly, D.J. Printech Press, Lubbock, TX, pp.

*Resources: Conservation and Use*, (Eds.) Callow, J.A. Ford*-*Loyd, B.V. and Newbury, H.J. Biotechnology in Agriculture Series, No. 19. CAB International, pp. 163–174. Adams, R.P., Miller, J.S., Golenberg, E.M. and Adams, J.E., 1994. *Conservation of Plant Genes* 

*11: Utilization of Ancient and Modern DNA*. Missouri Botanical Garden Press. St.

*Biotechnology of Spices, Medicinal and Aromatic Plants,* (Eds.) Edison, S., Ramana,

Havens 2009).

**7. References** 

156.

1–9.

new horizon to this field of applications.

Louis, MI, 276 pp. 227.

Biotechnology. Academic Press, New York.

(33.3; 72.1%). However, further studies should be initiated to improve regrowth of surviving embryogenic calluses and to study genetic stability after cryopreservation.
