**3. Methodologies**

## **3.1 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 Babu *et al*., 1997 can be used.

Murashige and Skoog (1962), Woody Plant (McCown and Amos, 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 (Nirmal Babu *et al*., 1997, 2005, Minoo 2002). These techniques form the base for establishing tissue cultures and developing *in vitro* technology for conservation. The basal media used are MS (Murshige and Skoog, 1962) for crops like cardamom, ginger, turmeric, kasturi turmeric, mango ginger, large cardamom, *Kaempferia*, *Vanilla* spp. seed and herbal spices and WPM-Woody Plant Medium (Mc Cown and Amos,

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

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

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 Babu *et al*., 1997 can be used. Murashige and Skoog (1962), Woody Plant (McCown and Amos, 1979) and Schenk and Hildebrandt (1972) media can be used depending upon the crop for micropropagation Table

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 (Nirmal Babu *et al*., 1997, 2005, Minoo 2002). These techniques form the base for establishing tissue cultures and developing *in vitro* technology for conservation. The basal media used are MS (Murshige and Skoog, 1962) for crops like cardamom, ginger, turmeric, kasturi turmeric, mango ginger, large cardamom, *Kaempferia*, *Vanilla* spp. seed and herbal spices and WPM-Woody Plant Medium (Mc Cown and Amos,

and DNA storage for overall objective of conserving gene pool.

rate of multiplication which in turn will reduce the extent of variation.

1. The miniaturized *in vitro* grown shoots can be used for cryopreservation.

**3. Methodologies 3.1 Micropropagation**  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

Table 1. Composition of MS\*, WPM\* and SH\* basal media

Protocols are available for micropropagation and multiplication of many endangerd species like *Piper hapnium, P. silent vallyensis, P.schmidtii, P. wightii, P. barberi , Vaniilla aphylla, V. pilifera, V. walkyrie, V. wightiana, K. rotunda and Alpinia galanga* are available (Peter et al 2002, Minoo 2002, Nirmal Babu et al 1999, 2005).

Bertaccini *et al* (2004), Du *et al (*2004) reported micropropagation and establishment of mitebrone virus-free garlic.

Cryopreservation of Spices Genetic Resources 461

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

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 0oC. 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

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

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

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,

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

**3.4 Pollen storage** 

few initial reports.

germplasm as pollen.

**3.5 Cryo preservation** 

1985; Kartha, 1985).

over 100 different plant species.

at cryogenic temperatures (Grout 1995).
