The production of whole plant by in vitro technique is an efficient, reliable and rapid strategy. This provides a faster method of large scale propagation. In vitro propagation in some wild and domestic Curcuma and Zingiber species of NE India was attempted with the aim of developing a general and reproducible protocol that could be used for further improvement and large scale plantation. In the present investigation, multiplication was found to occur by development of axillary buds which is ideal for maintaining genetic stability. However the rate of bud multiplication was significantly different according to the various concentrations and combinations of growth regulators used. Rate of multiplication was found to be dependent mainly on plant growth regulators, its concentration and nutrient media. The results demonstrated that for all the tissues used in the present study, MSR is more effective than MS media for obtaining a large number of plantlets in a short time.
The effect of different growth regulators and culture conditions on in vitro multiplication and rhizome formation was studied by Rout et al. (2001) for Z. officinale.
The importance of cytokinins has also been demonstrated for in vitro shoot production of other plants including Zingiberaceae (Chithra et al. 2005; Kesari et al. 2008). Cytokinins promote axillary shoot formation by inhibiting apical dominance. But the wild species with therapeutic value are investigated for the first time. In the current investigation, cytokinins along with lower concentrations of auxins revealed an increased proliferation and multiplication rate in Curcuma species. Explant type, growth medium, and endogenous hormone levels are known to influence shoot induction and these factors may contribute toward the variation observed in the present study. BA has been found to be more favourable than Kn in multiple shoot induction. However, persistence of explants in culture media containing higher concentration of cytokinins suppressed shoot elongation in present study which is contrary to what has been reported by other researchers who used rather high concentrations of plant growth regulators for the multiple shoot formation for some of the Zingiberaceae species (Khatun et al. 2003; Chan and Thong 2004, Bharalee et al. 2005, Sultana et al. 2009). In case of Zingiber species, the synergistic effect of the cytokinins was found fruitful. This was earlier found by many workers in other species of Zingiberaceae (Vincent et al. 1992; Nayak 2000; Khatun et al.
2003). However, Stanley and Keng (2007) used the combination of BA and IBA for Z.
zerumbet with much less number (2.3) of shoots per explant. But this was optimum for micropropagation when cultured in liquid medium. Fortunately the present study documents a better and convenient mode of in vitro propagation in the modified MS media with low percentage of agar. The semi-solid gel of agar proved to be beneficial for the regeneration and multiple bud formation in explants for all of the four species studied.
Sucrose is widely used as a standard carbon source for plant tissue culture, and different concentrations and different osmotic environments have been used. To the best of understanding till date, there are no reports on use of maltose as a carbon source in any members of Zingiberaceae. What is clear is that different carbon source is effective in supporting shoot multiplication probably due to the ability of this species to metabolize a wide variety of carbohydrates. Sugar at 2% concentration was also found to be satisfactory in micropropagation of Z. officinale earlier by Sharma and Singh (1995).
However higher concentration of sugar source has been found to be ideal for in vitro microrhizome production in Z. officinale (Sharma and Singh 1995; Zheng et al. 2008).
Increase in sugar concentration in the culture medium decreased the height of Curcuma plantlets significantly in current study. Hence, from the results of the study it can be concluded that maltose and sucrose are better carbon sources for in vitro micropropagation of the Zingiberaceae species in as low as 2% concentration.
The results obtained from RAPD profiling suggest that, in vitro regeneration and multiplication of C. longa, C. amada, Z. moran and Z. zerumbet using rhizome with axillary buds could be successfully used for rapid clonal propagation of these valuable medicinal plants with the least possibility of genetic variations. These can also be used as a source of disease free planting material for the farmers. The RAPD technique has been proved powerful in analyzing the genetic stability of regenerated plants in many other plant species (Rout and Das 2002; Lakshmanan et al. 2007). This is preferred over the other markers for cost effectiveness and simplicity and can be applied to rapid evaluation of genetic fidelity of micropropagated plants for the conservation of genetic richness.
Tissue culture techniques have been applied typically when traditional methods of propagation have either failed or proved inadequate. Plant tissue culture techniques have been increasingly applied to many medicinal plants in particular for mass propagation,
conservation of germplasm, study and production of bioactive compounds, and for genetic improvement. Medicinal plants have vast genetic diversity, which is a valuable source of agronomic gene/s of interest for the future. Large-scale plant tissue culture is found to be an attractive alternative approach to the traditional methods of plantations, as it offers a controlled supply of bio-chemicals independent of plant availability and more consistent product quality (Kesari et al. 2010). Application of in vitro propagation techniques may help in the conservation of biodiversity of locally used medicinal plants.
Therefore, as a principal output of this chapter, the protocols have been standardized for mass propagation of the valuable medicinal herbs, C. longa, C. amada, Z. moran and Z.
zerumbet through shoot morphogenesis, as well as cyto-genetic purity was established for the in vitro raised plants by RAPD, SDS-PAGE profiling and chromosome counting.
Present study is a successful effort to optimize the in vitro protocols for direct plantlet regeneration, rapid multiplication and effective conservation of wild and domestic species of Curcuma and Zingiber. It is interesting to note that till date, there is no any report on the endemic plant species Z. moran. The results will surely contribute for further research and conservation of wild and endemic varieties of Zingiberaceae that are available in limited quantities. The improved in vitro propagation protocol for the conservation of wild but medicinally sound species of Zingiber from NE India reported here could be helpful for the endemic species of Z. moran in terms of conservation, utilization and pharmaceutical application.