**4.4 Vanilla and related species**

Technology for cryopreservation of vanilla germplasm - using encapsulation and vitrification methods – were available. Encapsulated *in vitro* grown shoot tips of vanilla could be cryo preserved with 70% success when pretreated with progressive increase of sucrose concentration (0.1M-1.0M) for one day each and dehydrated for 8 hrs (Peter *et al* 2001; Minoo 2002 and Ravindran *et al* 2004) (Fig. 5).

Fig. 5. Germination of cryopreserved encapsulated shoot tips protocorms of vanilla

Ginzalez-Arnao, *et al*., (2009) attempted to cryo-preserve *V. planifolia* Andr. using *in vitro* fragmented explants (IFEs) and the apices derived from them. Cryopreservation of apices from *in vitro* grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of *in vitro* plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M

Cryopreservation of Ginger (*Zingiber officinale* Rosc) and turmeric (*Curcuma lo*nga L.) shoot tips was successfully done with 80% of recovery using vitrification method. But the rate of recovery was only 40% when encapsulated shoot tips were dehydrated in progressive increase of sucrose concentration together with 4- 8 hrs. of desiccation (Peter *et al* 2001 and

Efficient cryopreservation techniques were developed for *in vitro* grown shoots of ginger based on encapsulation dehydration, encapsulation vitrification and vitrification procedures (Yamuna et al 2007 and Yamuna 2007. The vitrification procedure resulted in higher regrowth (80 %) when compared to encapsulation vitrification (66 %) and encapsulation dehydration (41 %). The genetically stability of shoot apices was confirmed by molecular profiling. The RAPD and ISSR assays performed suggested that no genetic aberrations

Technology for cryopreservation of vanilla germplasm - using encapsulation and vitrification methods – were available. Encapsulated *in vitro* grown shoot tips of vanilla could be cryo preserved with 70% success when pretreated with progressive increase of sucrose concentration (0.1M-1.0M) for one day each and dehydrated for 8 hrs (Peter *et al*

Fig. 5. Germination of cryopreserved encapsulated shoot tips protocorms of vanilla

Ginzalez-Arnao, *et al*., (2009) attempted to cryo-preserve *V. planifolia* Andr. using *in vitro* fragmented explants (IFEs) and the apices derived from them. Cryopreservation of apices from *in vitro* grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of *in vitro* plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M

originated in ginger plants during culture and cryopreservation (Fig. 4).

**4.3 Ginger, turmeric and related species** 

Ravindran *et al* 2004).

**4.4 Vanilla and related species** 

2001; Minoo 2002 and Ravindran *et al* 2004) (Fig. 5).

sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature.

Minoo (2002) reported cryopreservation of vanilla pollen for conservation (Fig. 6) of haploid genome as well as assisted pollination between species that flower at different seasons and successful fertilisation using cryopreserved pollen (Minoo, 2002, Minoo *et al* 2011). Pollen from two asynchronously flowering species of *Vanilla viz*., cultivated *V. planifolia* and its wild relative *V. aphylla,* were cryopreserved after desiccation to 12 % moisture content, pretreated with cryoprotectant Dimethyl sulphoxide (5%) and cryopreserved -196ºC in Liquid Nitrogen. This cryopreserved pollen was latter thawed and tested for their viability both *in vitro* and *in vivo.* A germination percentage of 82.1% and 75.4% in *V. planifolia* and *V.aphylla* pollen respectively were observed indicating their viability(Fig.6). This cryopreserved pollen of *V. planifolia* was used successfully to pollinate *V.aphylla* flowers resulting in fruit set (Fig.7). The seeds thus obtaines were sussfully cultured to develop hybrid plantlets. This system is of great importance and can be used for conserving the haploid gene pool of *Vanilla* in cryobanks and their subsequent utility in crop improvement (Fig. 6 and 7)

Fig. 6. Germination of cryopreserved Vanilla pollen

Fig. 7. Fruit set after pollination with cryopreserved pollen

Cryopreservation of Spices Genetic Resources 475

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

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

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

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

encapsulated buds were hardened off and established in soil.

shoot tips from *in vitro*-grown plantlets of *S. francissi* germplasm.

primordia of hairy roots
