Secondary Metabolites of Medicinal Plants. Bharat Singh

culture medium supplemented with 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA). Growth kinetic studies were conducted by adopting settled cell volume and fresh/dry cell weight method. Two culture systems were used as growth medium (MS) and production medium (modified M9 medium) for production of cell biomass and naphthaquinones. The experimental results revealed that incubation under dark period enhanced the production of cell biomass and naphthaquinones. In case of production medium, absence of Na₂SO₄ was not effective in increasing the production naphthaquinones but increased the level of naphthaquinones production in air-lift bioreactor (Malik et al. 2008; Gupta et al. 2014). Similarly, a Box–Behnken statistical design was established as a bioreactor, which increased the production of acetyl shikonin in A. nobilis (Subramaniam et al. 2015). Increased naphthaquinones production was reported in culture media supplemented with KH₂PO₄ and K₂HPO₄ rather than NaH₂PO₄. In situ extraction resulted in significantly higher yield of naphthoquinone derivatives than control, along with higher volumetric and specific productivities. High-performance liquid chromatography (HPLC) analysis revealed that acetyl shikonin synthesized as the major naphthoquinone derivative than other compounds (Kumar et al. 2011; Malik et al. 2016). The nutritional factors like as phosphate supplementation in medium was observed as one of the most important nutritional factor affecting yield of both cell biomass and naphthaquinones (Saito 1993). The M9 medium containing KH₂PO₄ as the phosphate source increased on yield of naphthaquinones (Fujita et al. 1981a,b).

      The hairy root cultures provide an efficient method for increasing the production of shikonin in A. hispidissima. The nodal of plant material were infected with Agrobacterium rhizogenes followed for the development of hairy roots. The composition of medium was optimized for the induction of shikonin and found that RC medium showed rapid growth of cell biomass and induction of accumulation of shikonin. The production of shikonin was eightfold higher than control (Chaudhury and Pal 2010). The media combination was found to be the most suitable factor for induction of root cultures as well as shikonin production. Higher levels of IBA induced callusing from the roots. Some surprising results were also observed wherein roots were not submerged in the culture medium first, increased in length, and then started alkannin production. The synthesis alkannin occurred only in the upper surface of the roots tissue while synthesis was not reported in the inner surface in case of A. hispidissima (Bozan et al. 1999; Shekhawat 2012).

      Deoxyalkannin, alkannin, acetylalkannin, isobutyryl alkannin, β-hydroxyisovalerylalkannin, 2″-(S)-α-methylbutyryl alkannin, propionyl alkannin, teracrylalkannin, and acetyl shikonin were isolated from A. euchroma in vitro cultures. Similarly, methyl jasmonate and 1-monoglyceryl oleate, palmitate and stearate were also isolated from this genus (Damianakos et al. 2012). Arnebacene and arnebidin, along with arnebin-7 and vanillic acid, were isolated from the A. hispidissima roots (Ahmed et al. 2014). The cell culture medium of A. euchroma was elicited with phenylalanine to increase the production of shikonin, acetyl shikonin, and isobutyryl shikonin. Phenylalanine is considered as a key factor of phenylpropanoid pathway and found that it induced cell proliferation to promote the production of compounds. Phenylalanine was induced the cell biomass production up to 12-fold and production of compounds up to eightfold (Syklowska-Baranek et al. 2012a).

Elicitation studies of Rhizoctonia solani, methyl jasmonate, and salicylic acid were conducted for shikalkin production in A. euchroma callus. For the regeneration of callus, various types of explants (leaf, collar, and root) were inoculated onto LS medium. Maximum shikalkin biosynthesis was stimulated by White medium supplementing with indole-3-acetic acid (IAA) and kinetin. R. solani increased the pigment production by sevenfold higher than control. It is suggested that in polysaccharides of the fungal cells, a cascade of signaling induced the expression of genes those involved in the biosynthesis of shikalkin (Arghavani et al. 2015). Arnebia species in vitro culture research has been widely dedicated to improving shikonin production by application of various strategies, among them changing medium composition, addition of different elicitors, and various in situ extraction methods (Fu and Lu 1999; Ge et al. 2006; Chaudhury and Pal 2010; Kumar et al. 2011; Malik et al. 2011; Shekhawat and Shekhawat 2011).

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