Medicinal and aromatic plants form the essential components of biodiversity. There has been gradual and recently rapid loss of medicinal plant genetic resources because of many factors. At present 90% collections of medicinal plants are from the wild and the 70% of plants collections involve destructive harvesting (Mishra 2009). Also, majority of the medicinal plants come under herbaceous category and thereby easily harvested by people.
The family Zingiberaceae is one of the largest families comprising almost all the members with utmost medicinal values. It has 52 genera and about 1500 species distributed throughout tropical Asia especially in Indochina peninsula (Sirirugsa 1998). In NE India, the members of Zingiberaceae are found to grow naturally due to the favourable climate.
However, as a result of over exploitation, most of the members fall under the critically endangered or vulnerable category. Moreover, being rhizomatous the plants have a slower rate of propagation. Even the seed bearing members too are observed to propagate through rhizomes. Keeping in view all these factors, their demand in the pharma industry and traditional use, there is a strong need to increase the productivity and conservation of such plant species. But as these plants are herbaceous and propagate by vegetative means, there is less possibilities of hybrid production and cloning. Therefore, rapid multiplication through tissue culture techniques may definitely play a role in propagation and conservation of such important herbaceous plants. In vitro techniques could be used for conservation of medicinally significant members of Curcuma and Zingiber species, as proven in case of many Zingiberaceae species having condiment and spice value (Ashmore 1997).
In vitro culture technique has been recognized as an efficient tool for rapid clonal multiplication. Plant regeneration in vitro and re-introduction into the original or favourable habitats is one strategy for conservation of important plant species. Tissue culture is the technique of maintaining plant tissue in an artificial medium in vitro under controlled condition. Propagation of plants through tissue culture has become an important and popular technique. The continuous supply of sterile plantlets helps to overcome the contamination problem and reduce the time for sterilization process. Tissues from various organs such as stem and leaf of the axenic plantlets can be induced to form callus.
Moreover, callus tissue can serve as an experimental system to investigate the biological activities using specific bioassays. However, many factors contribute to the ability of a specific tissue to form callus such as medium and plant growth regulators. At present, researchers aim to produce substances with antitumor, antiviral, hypoglycemic, anti- inflammatory and antimicrobes through tissue culture technology. However, establishment of tissue culture system is required prior to further exploration of the biosynthetic capabilities of various in vitro cultures.
Monitoring the degree of genetic integrity between in vitro raised plants using molecular markers is desirable to reduce the chances of inclusion of variable genotypes.
Several strategies can be used to assess the genetic stability of in vitro derived plants such as karyological analysis, isozyme markers, DNA based markers etc. (Isabel et al. 1993).
Among different molecular markers used, random amplified polymorphic DNA (RAPD) is the cheapest and appears to be powerful tool for the detection of genetic variability and analysis of genetic fidelity of in vitro propagated plants and have been well established in many plants (Cassells et al. 1997; Salvi et al. 2001; Rout et al. 2001; Hussain et al. 2008).
Polypeptide profiling (SDS-PAGE) is a useful biochemical marker system with functional/expressed gene diversity and has been more popularly applied to seeds analysis for diagnostics and estimating out-crossing rates (Ferreira et al. 2000). The members of Zingiberaceae are known to show polyploidy in general. Therefore, checking the chromosome numbers of the regenerated plants could also establish the cytogenetic stability. The karyological study to check the ploidy status of in vitro grown plants was reported earlier by Panda et al. (2007).
Curcuma and Zingiber are two major genera of the family Zingiberaceae and used as one of the main cash crops in NE region. Besides some cultivated species of Curcuma and Zingiber, there are many other promising species from these two genera which are left unexplored. Many species also possess variety of medicinal properties and have been used by the tribal folk in traditional system of medicine over centuries. Such species need to be worked on. In fact, there are immense possibilities of growing such species commercially if in vitro tissue culture techniques can be applied. Cultivated species also await some better regeneration techniques to meet the demands and increase productivity. It will not only speed up the multiplication rate but also will enhance conservation and optimum utilization of the germplasm. Hence the present study is based on developing a better micropropagation technique in some wild and cultivated species from Curcuma (C. amada, C. longa) and Zingiber (Z. moran and Z. zerumbet) found in NE India leading to their in situ conservation and proper utilization.
The chapter has been divided into three objectives, first, micropropagation by direct organogenesis from axillary buds using various phytohormones individually and synergistically. The varying concentrations of cytokinins viz; Benzyl aminopurine (BA), Kinetin (Kn) alone and in combination with auxins viz, indole 3-buteric acid (IBA) and naphthalene acetic acid (NAA) were used for regeneration, multiplication and rooting response. Secondly, to establish optimum culture conditions to enhance the regeneration rate and mass multiply. The effect of two different growth media, Murashige and Skoog (MS) (Murashige and Skoog 1962) and a modified MS medium (MSR) along with varying concentrations of agar and carbon source was investigated for multiplying Curcuma and Zingiber species from rhizomatous bud. A comparison of initiation and multiplication of rhizome in MS media and MSR media with different hormone combination were also attempted in this study. The use of MSR media instead of liquid medium is a significant step in developing a standard protocol for rapid multiplication of species studied. The third objective was to produce a disease free material for conservation and further studies. In addition, we performed biomolecular techniques like RAPD analysis, SDS-PAGE analysis and karyological analysis of the micropropagated plants for cytogenetic purity assessment. The overall aim of the chapter was to develop
an efficient regeneration protocol from mature rhizomes for two species of Curcuma and Zingiber from NE India.