**3.4 Pollen storage**

460 Current Frontiers in Cryobiology

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 (Engelmann *et al* 1994). 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) post-

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.,* 1996; Venkataraman 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

Studies on conservation of cell lines is yet o become popular in spices. Suspensions of embryogenic cell lines of fennel, conserved at 4 0C for up to 12 weeks produced normal

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

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±2oC. 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

lavender (Ilahi and Jabeen, 1992; Ravindran *et al*, 1996; Sajina *et al*, 1997).

plants upon transfer to normal laboratory conditions (Umetsu *et al*, 1995).

**3.3 Somatic embryogenesis and plant regeneration** 

**3.2 Callus and cell culture systems** 

thaw treatment.

embryogenic cultures.

facilitate proliferation.

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 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 germplasm as pollen.
