MS, Murashige and Skoog medium; S, sucrose; M, mannitol; SC, screw cap; CP, cotton plug. aMean of 10 replications.

MS, Murashige and Skoog medium; S, sucrose; M, mannitol; SC, screw cap; CP, cotton plug. aMean of 10 replications.

Cell Suspension Culture

Plant cells accumulate secondary metabolites at specific cell sites at specific developmental stages under specific conditions of cell culture; hence, plant tissue culture can be used as an alternative to whole plants as a biological source of potentially useful metabolites and biologically active compounds (Yeoman, 1987). The genetic makeup, the regulation of gene expression, the cellular physiology, and the regulation of metabolism must be considered in order to develop highly productive and practical plant cell lines.

Production of volatile constituents in ginger cell cultures were reported earlier by Sakamura and Suga (1989). Ilahi and Jabeen (1992) also reported preliminary studies on alkaloid biosynthesis in callus cultures of ginger, and Charlwood et al. (1988) have reported the accumulation of flavor compounds by cultures of ginger. Nirmal Babu (1997) reported successful establishment of cell suspension cultures in ginger. These cultures are maintained by weekly transfers to the fresh nutrient medium containing MS basal salts and 1 mgl-1 of 2,4-D for over 2 years and are in continuous growth and multiplication. The cells were heterogeneous, and during the process of subcultures, some of the cells differentiated into oil-producing cells (see Figure 4.3f), although the number of oil-producing cells were less for commercial exploitation. These reports are very preliminary and much more work needs to be done before ginger cell cultures can be used for commercial production of flavor components in vitro.

Protoplast Isolation and Culture

Successful isolation, culture, and fusion of protoplasts are important because of their role in studies of plant improvement by cell modification and somatic hybridization. Another aspect of considerable interest is the storage of protoplast through immobilization and cryopreservation, which are of great importance, especially in the pharmaceutical industry (Bajaj, 1989a, 1989b).

Protoplasts were successfully isolated from young in vitro—derived leaves (see Figure 4.3f) using an enzyme mixture containing 0.5 percent macerozyme R10, 3 percent hemicellulase, and 5 percent cellulase Onozuka R10 and mechanically macerating the plamolysed leaf tissue after incubating at 15°C for 10 hours and at 30°C for 6 hours. The protoplast yield was 2.5 X 105 per gram of leaf tissue with 55 percent viability. The isolated protoplasts were round and were filled with chloroplasts. Cell suspension cultures required a slightly different concentration of enzyme mixture with 1 percent macerozyme R10, 3 percent hemicellulase and 6 percent Onozuka cellulase R10. The incubation conditions were 15°C for 10 hours and 30°C for 8 hours. The yield of protoplasts was lesser from a callus/cell suspension with 1 X 105 protoplasts per gram weight of callus in an isolation solution containing cell protoplast washing (CPW) salts, 7 percent mannitol, 1 percent macerozyme, 3 percent hemicellulase, and 6 percent cellulase Onozuka R10. The protoplasts isolated from cell suspension cultures were round, with little or no chloroplasts inside, with 72 percent viability (see Table 4.10). The protoplasts derived from leaf tissue were heterogeneous and comprised of protoplasts of different sizes (0.15 to 0.21 mm). The protoplasts derived from cell suspension cultures were mostly of uniform size (0.39 mm). They were cultured up to 20 days as droplet cultures in MS liquid medium with 0.5 mgl-1 BAP, 0.5 mgl-1 NAA, and 0.5 mgl-1 2,4-D supplemented with 3 percent sucrose and 7 percent mannitol. The cell contents became dense by 1 week and fresh medium needed to be added at 7-day intervals. The protoplasts started regenerating the cell wall within 2 to 3 days. Within 3 to 4 weeks of culture, cells started dividing. Protoplasts plated on liquid as well as solidified medium

Table 4.10 Effect of source tissue, enzyme concentration, and incubation conditions on yield of protoplasts

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