**4.2 Cardamom and related species**

Choudhary and Chandel (1995) attempted cryo-conservation of cardamom (*Elettaria cardamomum* Maton.) seed. They tried to conserve seeds at ultra-low temperature by suspending seeds in cryovials in vapor phase of liquid nitrogen (-150oC) by slow freezing and also by direct immersion in liquid nitrogen (-196oC) by fast freezing. The result showed that seeds possessing 7.7-14.3% moisture content could be successfully cryo-preserved with 80% germination when tested after one-year storage in vapor phase of liquid nitrogen (at-150oC).

Shoot tips(1.0-2.0mm) from *in vitro* grown plantlets of cardamom were subjected to progressive increase of sucrose concentrations (0.1, 0.3, 0.5, 0.7, 0.9, and 1.0) for two days each under the same cultural conditions as the parent plantlets. These shoot tips were transferred to 1.8ml cryotube containing ice cold PVS2 solution (30%(v/v) glycerol + 15% (v/v) ethylene glycol + 15% (v/v) DMSO in culture medium with 0.4 M sucrose, pH (5.8) ) at 00C for 3 hours. After 3 hours equilibration at 00C, the shoot tips were directly immersed into liquid nitrogen for 1 hour. Vials were thawed in 400C water for 1 minute. The cryoprotectant was removed and the shoot tips were washed 2-3 times in 1.2M sucrose solution. About 70%Shoot tips were recovered on MS medium supplemented with BAP and NAA. But the encapsulation vitrification method gave only 60% success (Ravindran *et al*  2004).

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

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

Choudhary and Chandel (1995) attempted cryo-conservation of cardamom (*Elettaria cardamomum* Maton.) seed. They tried to conserve seeds at ultra-low temperature by suspending seeds in cryovials in vapor phase of liquid nitrogen (-150oC) by slow freezing and also by direct immersion in liquid nitrogen (-196oC) by fast freezing. The result showed that seeds possessing 7.7-14.3% moisture content could be successfully cryo-preserved with 80% germination when tested after one-year storage in vapor phase of liquid nitrogen (at-

Shoot tips(1.0-2.0mm) from *in vitro* grown plantlets of cardamom were subjected to progressive increase of sucrose concentrations (0.1, 0.3, 0.5, 0.7, 0.9, and 1.0) for two days each under the same cultural conditions as the parent plantlets. These shoot tips were transferred to 1.8ml cryotube containing ice cold PVS2 solution (30%(v/v) glycerol + 15% (v/v) ethylene glycol + 15% (v/v) DMSO in culture medium with 0.4 M sucrose, pH (5.8) ) at 00C for 3 hours. After 3 hours equilibration at 00C, the shoot tips were directly immersed into liquid nitrogen for 1 hour. Vials were thawed in 400C water for 1 minute. The cryoprotectant was removed and the shoot tips were washed 2-3 times in 1.2M sucrose solution. About 70%Shoot tips were recovered on MS medium supplemented with BAP and NAA. But the encapsulation vitrification method gave only 60% success (Ravindran *et al* 

*Piper barberi* was reported by Peter *et al* (2001) and Ravindran *et al* (2004).

preservation of *P.barberi* propagules.

**4.2 Cardamom and related species** 

150oC).

2004).

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

Cryopreservation of Spices Genetic Resources 471

dehydrated with PVS2 for 60 min retained a high level of shoot formation (70 %). The vitrification procedure resulted in higher regrowth (70 %) (Fig.3) when compared to encapsulation vitrification (62 %) and encapsulation dehydration (60 %). In all the three cryopreservation procedures tested, shoots grew after cryopreservation without intermediary callus formation. The genetic stability of cryopreserved cardamom shoots were

Fig. 4. Plant regeneration from cryopreserved shoot buds of ginger by encapsulation

& d) Shoot buds encapsulated in Na-alginate, e) & f) Shoot buds turned brown after thawing, g) Viable apical dome stained in red colour after liquid nitrogen storage (TTC staining), h) Regenerating shoot bud 20 days after post culturing, i) & j) Elongated shoot with no intermediary callus formation, k) & l) Regenerating shoot buds in petriplates, m)

Plantlets regenerating from cryopreserved shoot bud

vitrification. a) *In vitro* culture, b) A typically excised shoot bud used for cryopreservation, c)

confirmed using ISSR and RAPD profiling.

Yamuna (2007) tested the effect of encapsulation – dehydration, encapsulation vitrification and vitrification methods on cryopreservation of cardamom. In the vitrification treatment, to enhance tolerance to vitrification solution (PVS2), a two step sucrose preculture with 0.3 M and 0.75 M sucrose for one day each and an osmo protection step with a loading solution (LS) of 2 M glycerol and 0.4 M sucrose were performed prior to PVS2 treatment. The shoots

Fig. 3. Plant regeneration from cryopreserved miniature shoots of cardamom by vitrification. a) Cardamom culture with miniature shoots, b) & c) Excised meristematic clumps used for cryopreservation, d) Explant turned brown after cryopreservation, e) Viable tissues stained in TTC after cryopreservation, f), g), h), & i) Shoot development after 10, 14 and 25 days of post culturing , j) regenerating shoot buds in a petridish, k) Development of multiple shoots after 4 months of post culturing

Yamuna (2007) tested the effect of encapsulation – dehydration, encapsulation vitrification and vitrification methods on cryopreservation of cardamom. In the vitrification treatment, to enhance tolerance to vitrification solution (PVS2), a two step sucrose preculture with 0.3 M and 0.75 M sucrose for one day each and an osmo protection step with a loading solution (LS) of 2 M glycerol and 0.4 M sucrose were performed prior to PVS2 treatment. The shoots

Fig. 3. Plant regeneration from cryopreserved miniature shoots of cardamom by vitrification. a) Cardamom culture with miniature shoots, b) & c) Excised meristematic clumps used for cryopreservation, d) Explant turned brown after cryopreservation, e) Viable tissues stained in TTC after cryopreservation, f), g), h), & i) Shoot development after 10, 14 and 25 days of post culturing , j) regenerating shoot buds in a petridish, k) Development of

multiple shoots after 4 months of post culturing

dehydrated with PVS2 for 60 min retained a high level of shoot formation (70 %). The vitrification procedure resulted in higher regrowth (70 %) (Fig.3) when compared to encapsulation vitrification (62 %) and encapsulation dehydration (60 %). In all the three cryopreservation procedures tested, shoots grew after cryopreservation without intermediary callus formation. The genetic stability of cryopreserved cardamom shoots were confirmed using ISSR and RAPD profiling.

Fig. 4. Plant regeneration from cryopreserved shoot buds of ginger by encapsulation vitrification. a) *In vitro* culture, b) A typically excised shoot bud used for cryopreservation, c) & d) Shoot buds encapsulated in Na-alginate, e) & f) Shoot buds turned brown after thawing, g) Viable apical dome stained in red colour after liquid nitrogen storage (TTC staining), h) Regenerating shoot bud 20 days after post culturing, i) & j) Elongated shoot with no intermediary callus formation, k) & l) Regenerating shoot buds in petriplates, m) Plantlets regenerating from cryopreserved shoot bud

Cryopreservation of Spices Genetic Resources 473

sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to

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

plant vitrification solution PVS3 for 30 min at room temperature.

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
