## Introduction
### Chemical constituents
#### Pharmacological activities of withanolides
MS liquid medium containing 1.0 mg BA/L and 10% coconut milk favoured a maximum increase in biomass (27 fold), induced microshoots (37.6) as well as accumulation of withaferin A (0.14%). Direct rooting from leaf explants of _W. somifera_ has been achieved on half-strength MS medium supplemented with 15 g/l sucrose, and different concentrations of growth regulators [76]. The roots were cultured on MS liquid medium for the establishment of root-organ cultures with the same plant growth regulators and incubated on an orbital shaker at 80 rpm at 25\(\pm\)2 'C. The concentration of alkaloids increased compared to field grown roots. The maximum concentration of withanolides (10 mg/g dry weight) was obtained in the bioreactor. Recently, withanolide A biogeneration in shoot cultures of _W. somifera_ has been reported [77]. Multiple shoot cultures of two experimental lines of _W. somifera_ were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin influenced morphogenetic response as well as differentially modulating the level of biogeneration of withanolide A in the _in vitro_ shoots of the two lines. The production of withanolide A in the cultures varied considerably (_ca._ 10-fold, 0.014 to 0.14 mg/g fresh weight) according to the hormone composition of the culture media as well as the genotype used as the explant source. The shoot culture of experimental lines cultivated at 1.0 ppm of BAP and 0.5 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 %. **Hairy root cultures as a source of withanolides**  
In the two last decades, the hairy root system based on _Agrobacteriumrium rhizogenes_ inoculation has become popular as a method of producing secondary metabolites synthesized in plant roots [78-79]. Unorganized plant tissue cultures are frequently unable to produce secondary metabolites at the same levels as the intact plant. The hairy root phenotype is characterized by fast, hormone-independent growth, lack of geotropism, lateral branching and genetic stability. The secondary metabolitesproduced by hairy roots arising from the infection of plant material by _A. rhizogenes_ are the same as those usually synthesized in intact parent roots, with similar or higher yields [80]. This feature, together with genetic stability and generally rapid growth in simple media without phytohormones, makes them especially suitable for biochemical studies not easily undertaken with root cultures of an intact plant. [PERSON] _et al_. [81] carried out hairy root transformation of _W. somifera_ by three different strains of _A. rhizogenes_ (A4, LBA 9402 and LBA 9360) and analyzed the specificity and frequency of their withhanolide production with special reference to withaferin A. The best response in terms of transformation ability and growth of the hairy roots was obtained with strain A4, followed by LBA 9402; LBA 9360 failed to induce a transformation event. The production of withaferin A was studied in the A4-induced hairy root lines at different growth phases (4, 10 and 24 weeks) using HPLC (high performance liquid chromatography) and maximum levels were observed in the media and hairy roots of 10-week-old cultures. During the infection process _A. rhizogenes_ transfers a part of the DNA (transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant cells and the genes contained in this segment are expressed in the same way as the endogenous genes of the plant cells [82]. Some _A. rhizogenes_, such as strain A4, have the T-DNA divided in two sections, the TR-DNA and TL-DNA, each of which can be incorporated separately into the plant genome. Two sets of pRi genes are involved in the root induction process: the _aux_ genes located in the TR region of the pRi T-DNA and the _rol_ (root loci) genes of the TL region [83]. The _ags_ genes responsible for opine biosynthesis in the transformed tissues are also located in the TR region [84].