Cryopreservation of Spices Genetic Resources

Plant genetic resources constituting genotypes or populations of cultivars (landraces, advance/improved cultivars), genetic stocks, wild and weedy species, which are maintained in the form of plants, seeds, tissues, etc. hold key to food security and sustainable agricultural development (Iwananga, 1994). They are non-renewable and are among the most essential of the world’s natural resources. Due to deforestation, spread of superior varieties and selection pressure, genetic variability is gradually getting eroded. This demands priority action to conserve germplasm be it at species, genepool or ecosystem level, for posterity (Frankel, 1975).


Introduction
Plant genetic resources -constituting genotypes or populations of cultivars (landraces, advance/improved cultivars), genetic stocks, wild and weedy species, which are maintained in the form of plants, seeds, tissues, etc. -hold key to food security and sustainable agricultural development (Iwananga, 1994). They are non-renewable and are among the most essential of the world's natural resources. Due to deforestation, spread of superior varieties and selection pressure, genetic variability is gradually getting eroded. This demands priority action to conserve germplasm be it at species, genepool or ecosystem level, for posterity (Frankel, 1975).
Whilst ecologists focused on in situ conservation might argue that ex situ conserved germplasm cannot offer the advantages afforded by selection and adaptation as a result of environmental pressures, there is no denying that if species are under threat-or worse, near extinction-then ex situ conservation of even limited germplasm is preferable to extinction. The opportunities offered by conservation biotechnology should not be missed or restricted by lack of interconnectivity between traditional and contemporary conservation practitioners.

Spices and germplasm conservation
Spices and herbs are aromatic plants-fresh or dried plant parts like foliage, young shoots, roots, bark, buds, seeds, berries and other fruits of which are mainly used to flavour our culinary preparations, confectionary. They are also major ingredients in indigenous medicine and perfumery. Spices and herbs are grown throughout the world-different plant species in different regions. Peninsular India is a rich repository of spices and over 100 species of spices and herbs are grown. The other major spice growing countries are Brazil, China, Guatemala, Indonesia, Madagascar, Nigeria, West Indies, Malaysia, Sri Lanka, Spain, Turkey, Mediterranean region and the Central America. Black pepper, cardamom, ginger, turmeric, vanilla, capsicum, cinnamon, clove, nutmeg, tamarind, coriander, cumin, fennel, fenugreek, dill, caraway, anise and herbs like saffron, lavender, thyme, oregano, celery, anise, sage and basil are important as spices. India being the native home of many spices, their conservation and characterization are one of the priority programmes. Deforestation, habitat degradation and overexploitation caused considerable loss of diversity in spices.

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In many spices, conventional seed storage can satisfy most of the conservation requirements. But in crops with recalcitrant seeds and those having conservation needs cannot be satisfied by seed storage, have to be stored in vitro. Most field gene banks are prone to high labour cost, vulnerable to hazards like natural disasters, pests and pathogens attack (especially viruses and systemic pathogens), to which they are continuously exposed and require large areas of space. This supports in vitro and cryo conservation. In addition, other resources like continuous supply of standard stock cultures for experiments to examine physiological and biochemical processes, cell and callus lines developed for in vitro synthesis of valuable secondary products, flavours and other important compounds will benefit strongly from in vitro cultures. Most of the spice crops are either vegetatively propagated or have recalcitrant seeds. The spices germplasm is mostly conserved in field gene banks. Most of the spices are plagued by destructive and epidemic diseases caused by viruses, bacteria and fungi. This makes germplasm conservation in field gene bank risky. Thus in vitro and cryo storage system becomes important in the overall strategy of conserving genepool. Each technology should be chosen on the basis of utility, security and complementarily to other components of the strategy. A balance needs to be struck between seed, field gene bank, in vitro and cryo conservation of propagules, tissues, pollen, cell lines and DNA storage for overall objective of conserving gene pool.

Micropropagation
Plant regeneration and successful cloning of genetically stable plantlets in tissue culture is an important pre-requisite in any conservation effort of recalcitrant species. These techniques form the base for establishing tissue cultures and developing in vitro and cryo conservation technology for conservation. Simultaneously these tissue-cultured plants should be evaluated for their morphological and genetic stability in culture. The in vitro storage experiments, as much as possible, use growth regulators free media to reduce the rate of multiplication which in turn will reduce the extent of variation.
Micropropagation (culture initiation, multiplication, plant regeneration and in vitro rooting) form the cycle of events that form the backbone of cryopreservation studies. For initial culture establishment earlier protocols developed by Nirmal  can be used. Murashige andSkoog (1962), Woody Plant (McCown andAmos, 1979) and Schenk and Hildebrandt (1972) media can be used depending upon the crop for micropropagation Table  1. The miniaturized in vitro grown shoots can be used for cryopreservation.
Micropropagation protocols for stable cloning of elite genotypes of spice crops were standerdised. Protocols were available for black pepper and its related species cardamom, ginger, turmeric and related genera, large cardamom, kasturi turmeric, mango ginger, Kaempferia galanga, K. rotunda, Alpinia spp, large. Cardamom, vanilla and related species, cinnamon, camphor, cassia seed and herbal spices like lavender, celery, thyme, mint, anise, savory, spearmint and oregano , Minoo 2002 1979) for black pepper and its related species, cinnamon, camphor and cassia. Simultaneously these tissue-cultured plants are being evaluated for their morphological and genetic stability in culture (Luckose et al, 1993, Chandrappa et al, 1997, Nirmal babu et al 2003, Madhusoodanan et al 2005. Though micropropagation protocols were standardized using growth regulators, all the in vitro storage experiments were carried out using growth regulators free media to reduce the rate of multiplication which in turn will reduce the extent of variation.  *Murashige and Skoog, 1962, McCown and Amos, 1979, Schenk and Hildebrandt 1972 (Peter et al 2002, Minoo 2002, Nirmal Babu et al 1999. Bertaccini et al (2004), Du et al (2004) reported micropropagation and establishment of mitebrone virus-free garlic.

Composition
www.intechopen.com Quatrano (1968) and Nag and Street (1973) reported the first successful experiments on cryopreservation of plant cells. Since then a large number of cell suspension and calli cultures have been successfully cryopreserved . In general, callus cultures are more difficult to cryopreserve than cell suspensions, because of the relative volume of the callus, its slow growth rate and the cellular heterogeneity (Withers 1987). One successful cryopreservation procedure that is applicable to all different cell suspensions or calli cultures has not been developed yet. Research focuses on optimizing the factors on which successful cryopreservation of plant organs cells suspensions and calli depends, such as: (i) starting material, (ii) pretreatment, (iii) cryopreservation procedure, and (iv) postthaw treatment.

Callus and cell culture systems
Plant cells cultured in vitro produce wide range of primary and secondary metabolites of economic value. Production of phytochemicals from plant cell cultures has been presently used for pharmaceutical products. Production of flavour components and secondary metabolites in vitro using immobilised cells is an ideal system for spices crops. Production of saffron and capsaicin was reported using such system (Ravishankar et al., 1988;1993, Johnson et al., 1996Venkataraman and Ravishankar 1997). Johnson et al (1996) reported biotransformation of ferulic acid vanillamine to capsacin and vanillin in immobilised cell cultures of Capsicum frutescens. Reports on the in vitro synthesis of crocin, picrocrocin and safranel from saffron stigma (Himeno and Sano, 1995) and colour components from cells derived from pistils (Hori et al, 1988) are available for further scaling up. Callus and cell cultures were established in nutmeg, clove, camphor, ginger, lavender, mint, thyme, celery etc. Cell immobilization techniques have been standardized in ginger, sage, anise and lavender (Ilahi and Jabeen, 1992;Ravindran et al, 1996;Sajina et al, 1997).
Studies on conservation of cell lines is yet o become popular in spices. Suspensions of embryogenic cell lines of fennel, conserved at 4 0 C for up to 12 weeks produced normal plants upon transfer to normal laboratory conditions (Umetsu et al, 1995).

Somatic embryogenesis and plant regeneration
In black pepper primary embryogenic cultures can be established as per the method described by Nair and Dutta Gupta (2003). Culture the surface sterilized seeds on agar gelled full-strength, PGR-free SH (Schenk and Hildebrandt, 1972) medium containing 3.0% (W/V) sucrose under darkness. Primary somatic embryos (PEs) derived from micropylar tissues of germinating seeds after 90 days could be utilized for inducing secondary somatic embryogenic cultures.
Primary somatic embryo clumps having pre-globular to torpedo shaped embryos (5-6 visible embryos per seed) were carefully detached and inoculated on half strength PGR-free SH medium containing 1.5 % sucrose and gelled with 0.8% agar (Bacteriological grade, Himedia). The pH of the medium was adjusted to 5.9 prior to autoclaving. Cultures were maintained at darkness at a temperature of 25±2 o C. The culture conditions remained the same for all further experiments unless otherwise specified. While inoculating, the PEs were uniformly spread on the surface of the medium. Secondary embryogenic cultures were further maintained by subculturing on SH medium containing 1.5% sucrose at intervals of 20 d. The proliferating SEs were spread periodically on the surface of the medium, to facilitate proliferation.

Pollen storage
Pollen storage can be considerable value supplementing the germplasm conservation strategy by facilitating hybridisation between plants with different time of flowering and to transport pollen across the globe for various crop improvement programmes in addition to developing haploid or homozygous lines. No significant work was done in India, except a few initial reports.
The technique of pollen storage is comparable with that of seed storage, since pollen can be dried (less than 5% moisture content on a dry weight basis) and stored below 0 o C. There are limited reports on the survival and fertilizing capacity of cryopreserved pollen more than five years old. Pollen might represent an interesting alternative for the long-term conservation of problematic species (IPGRI, 1996). However, pollen has a relatively short life compared with seeds (although this varies significantly among species) and viability testing can be time-consuming and uneconomical. Other disadvantages of pollen storage are the small amount produced by many species, the lack of transmission of organelle genomes via pollen, the loss of sex-linked genes in dioecious species and the general inability to regenerate into plants. Pollen, therefore, has been used to a limited extent in germplasm conservation (Hoekstra ,1995). An advantage is that pests and diseases are rarely transferred by pollen (excepting some virus diseases). This allows safe movement and exchange of germplasm as pollen.

Cryo preservation
For long-term conservation of the problem species, cryopreservation is the only method currently available. Dramatic progress has been made in recent years in the development of new cryopreservation techniques and cryopreservation protocols have been established for over 100 different plant species.
Cryopreservation is an attractive option for long-term storage. Liquid nitrogen (-196°C) is routinely used for cryogenic storage, since it is relatively cheap and safe, requires little maintenance and is widely available. Below -120°C the rate of chemical or biophysical reactions is too slow to cause biological deterioration (Kartha 1985). Only in the long term might there be a small risk of ionising radiation causing genetic changes in materials stored at cryogenic temperatures (Grout 1995).
An array of plant material could be considered for cryopreservation as dictated by the actual needs vis-a-vis preservation. These include meristems, cell, callus and protoplast cultures, somatic and zygotic embryos, anthers, pollen or microspores and whole seeds (Withers, 1985;Kartha, 1985).
Plant germplasm stored in liquid nitrogen (-196°C) does not undergo cellular divisions. In addition, metabolic and most physical processes are stopped at this temperature. As such, plants can be stored for very long time periods and both the problem of genetic instability and the risk of loosing accessions due to contamination or human error during subculturing are overcome. Most cryopreservation endeavours deal with recalcitrant seeds, in vitro tissues from vegetatively propagated crops, species with a particular gene combination (elite genotypes) and dedifferentiated plant cell cultures. Care must be taken to avoid ice crystallisation during the freezing process, which otherwise would cause physical damage www.intechopen.com 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. However, their routine utilisation is still restricted almost exclusively to the conservation of cell lines in research laboratories For small volumes, long-term storage is practicable through storage of cultures in cryopreservation at ultra-low temperature, usually by using liquid nitrogen (-196 o C). 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 (-196 0 C ) c a n b e a c h i e v e d b y d i f f e r e n t t e c h n i q u e s l i k e d i r e c t f r e e z i n g , encapsulation-dehydration, encapsulation-vitrification and vitrification.

Encapsulation -Dehydration
A simplified methodology for vitrification is given below (Yamuna 2007).
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 which made them suitable for cryopreservation.
1. Suspend in vitro grown shoots/ somatic embryos in MS basal medium supplemented with 4% (w/v) Na alginate, 2M Glycerol and 0.4 M sucrose. 2. Drop the mixturecontining microshoots, with a sterile pipette into 0.1M CaCl 2 solution containing 2M Glycerol and 0.4M sucrose and left for 20 min to form beads about 4 mm in diameter, each bead containing at least one shoot. 3. Preculture the encapsulated shoots -stepwise -on MS medium enriched with different concentration of 0.3, 0.5, 0.75 and 1.0M for four days with one day on each. 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 periods of 0-10 h to determine the optimal dehydration time. 5. Measure the water content of the beads was by weighing them prior and after drying in an oven at 80 0 C for 48h. 6. Transfer the dehydrated beads into a 2 ml cryovial (ten beads per tube) and directly immerse in liquid nitrogen for 24h.

Vitrification
A simplified methodology for vitrification is given below (Yamuna 2007).
2. The treated explants were then cultured on MS medium supplemented with sucrose at 0.75 M for 1 day in the same conditions. 3. After pretreatments explants were transferred to a cryovial with 1.8 ml of loading solution (2 M Glycerol + 0.4 M sucrose) and kept for 15 min. 4. Different incubation periods in PVS2 (40-100 minutes) were tested for osmoprotected explants 5. Cryovials containing 8-10 explants were directly immersed in liquid nitrogen and kept for 24 h.
1. Suspend pre-cultured shoots (1-2mm)/ somatic embryos with 2-3 apical domes on 0.3M sucrose for 16h in MS basal medium supplemented with 4% sodium alginate and 0.3 M sucrose. 2. Dispense the mixture including shoots, were with a sterile pipette into MS medium supplemented with 0.1M CaCl 2 and 0.4 to 1.0M sucrose, with or without 2M Glycerol gently shaken (20 rpm) on a rotary shaker for 1h at 25 0 C. 3. The encapsulated and osmo-protected shoots were dehydrated with 20 ml PVS2 in a 100 ml Erlenmeyer flask at 25 0 C and plunged into LN and held for at least 24 h at -196 0 C.

Thawing and recovery of conserved materials
After LN storage, cryovials warm rapidly in a 40 0 C 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.

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 vitro 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.
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., 1995or 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.

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

Black pepper and related species
Cryopreservation of black pepper (Piper nigrum L.)seeds in liquid nitrogen (LN 2 ) 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 -196 0 C, with a survival rate of 45% & 10.5% respectively (Chaudhury and Chandel 1994).
www.intechopen.com  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 www.intechopen.com 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 25 0 C 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 0 0 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 0 C) and kept for one hour (Peter et al 2001 andRavindran et al 2004). Yamuna 2007 also reported that studies on cryopreservation of endangered P.barberi s h o o t t i p s r e v e a l e d t h a t , t h e 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. 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 (-150 o C) by slow freezing and also by direct immersion in liquid nitrogen (-196 o C) 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-150 o C).

Cardamom and related species
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 0 0 C for 3 hours. After 3 hours equilibration at 0 0 C, the shoot tips were directly immersed into liquid nitrogen for 1 hour. Vials were thawed in 40 0 C 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).
www.intechopen.com 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.

Ginger, turmeric and related species
Cryopreservation of Ginger (Zingiber officinale Rosc) and turmeric (Curcuma longa 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 andRavindran et al 2004).
Efficient cryopreservation techniques were developed for in vitro grown shoots of ginger based on encapsulation dehydration, encapsulation vitrification and vitrification procedures 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 originated in ginger plants during culture and cryopreservation (Fig. 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 andRavindran et al 2004) (Fig. 5).  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

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 al 2003, 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.  (Phunchindawan et al., 1997). Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M 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 0.5M sucrose and 1M or 1.5M 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

Reports of cryoconservation of spices like
The effect of sucrose concentration and dehydration period on survival and regrowth of encapsulated calluses were also studied in 2 species of Crocus

DNA bank
Concurrent with the advancements in gene cloning and transfer has been the development of technology for the removal and analysis of DNA. DNAs from the nucleus, mitochondrion and chloroplast are now routinely extracted and immobilized onto nitrocellulose sheets where the DNA can be probed with numerous cloned genes. In addition, the rapid development of polymerase chain reaction (PCR) now means that one can routinely amplify specific oligonucleotides or genes from the entire mixture of genomic DNA.These advances, coupled with the prospect of the loss of significant plant genetic resources throughout the world,have led to the establishment of DNA bank for the storage of genomic DNA.
The conserved DNA will have numerous uses viz, molecular phylogenetics and systematics of extinct taxa, production of previously characterized secondary compounds in transgenic cell cultures, production of transgenic plants using genes from gene families, in vitro expression and study of enzyme structure and function and genomic probes for research laboratories.
The vast resources of dried specimens in the world's herbaria may hold considerable DNA that would be suitable for PCR. It seems likely that the integrity of DNA would decrease with the age of specimens. Because there are many types of herbarium storage environments, preservation and collections, there is a need for systematic investigations of the effect of modes of preparation, collection and storage on the integrity of DNA in the world's major holdings.
The advantage of storing DNA is that it is efficient and simple and overcomes many physical limitations and constraints that characterize other forms of storage (Adams 1988, 1997, Adams and Adams 1991, Adams et al 1994. The disadvantage lies in problems with subsequent gene isolation, cloning and transfer but, most importantly, it does not allow the regeneration of live organisms (Maxted et al., 1997). DNA banking is yet to catch up in spices. DNA samples of over 600 genotypes of spices is stored in the DNA bank of Indian Institute of Spices Research (IISR), Calicut.

Future focus
In contrast to the prevailing attitude among conservation biologists, globally there is considerable interest among cryobiologists in the use of in vitro, cold and ultra-cold technology for germplasm conservation. The procedures for plant material are given indepth coverage by Reed et al. (2004) who stress equally the ecological and plant/germplasm health aspects preceeding and following storage. Panis and Lambardi (2006) discussed the evolution of technologies for plant material, covering cell suspensions and callus cultures of herbaceous species, pollen, shoot meristems, woody species, as well as seed and embryonic axes. The ex situ gene bank at Gatersleben in Germany houses 986 potato accessions are cryopreserved and trials on other species are performed (Börner 2006). The National Bureau of Plant Genetic Resources (NBPGR), New Delhi has over 702 accesion of various spices, medicinal and aromatic crops in its cryo gene bank (Table. 3). Keller et al. (2008) make the point that cryopreservation affords the best of conditions for the long-term maintenance of www.intechopen.com 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 Havens 2009).
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 new horizon to this field of applications.