2.4.1. In vitro production
Various alternative techniques for the commercial production of PPT depend on the availability of cost-effective raw materials. In this regard, the callus cultures of P. peltatum shows a higher accumulation of PPT in Murashige and Skoog’s (MS) medium supplemented with kinetin and 2, 4-dichloro phenoxy acetic acid. Chromatographic analysis of PPT by thin-layer chromatography (TLC) identified two factors, age of callus and concentration of the hormone in medium, respectively, which control the in vitro production of PPT (Kadkade, 1981). Analysis of the metabolites by HPLC coupled to tandem mass spectrometry analysis showed incorporation of 3,4-methylenedioxy cinnamic acid (MDCA) in feeding experiments in cell culture of L. album , and DOP appeared to be a rate-limiting point in PPT biosynthesis (Seidel et al., 2002).
An alternative source of PPT from P. peltatum can be achieved by the use of embryogenic callus and adventitious root culture techniques (Anbazhagan et al., 2008). The addition of 2,4-dichloro phenoxy acetic acid (2,4-D) in embryogenic callus and indole-3-butyric acid in adventitious root callus trigger the PPT production in MS medium. High-performance liquid chromatography (HPLC) analysis showed that a higher amount of PPT was obtained from adventitious roots than embryogenic cell clumps of P. peltatum . Still, potentially, both approaches can efficiently be used to produce PPT on a large-scale via the use of bioreactors (Anbazhagan et al., 2008). Podophyllotoxin production from P. hexandrum adventitious root can also be optimized by varying concentrations of sucrose, ammonium, phosphate, and pH of the nutrient medium. Podophyllotoxin accumulation was reported to be highest (6.4 mg/g dry weight, DW) at 6% sucrose, 10mM ammonium, 2.25mM phosphate, and pH 6.0 in the optimized MS medium (Rajesh et al., 2014).
An alternative hairy root culture of L. narbonense for the production of PPT yielded five-fold higher levels of justicidin B (7.78mg/g, DW) compared to callus. The content of justicidin B in theL. narbonense intact roots is about 0.5mg/g DW, in callus cultures is 1.57mg/g DW. To meet the industrial requirement and large-scale production of plant anticancer agent justicidin B fromL. narbonense, hairy roots bioreactor system “BIOSTAT B plus” can be a feasible alternative. Compared to callus, fivefold higher yields of justiidin B (7.78 mg/g DW) can be produced from hairy roots by this technique (Ionkova et al., 2013). A cross-species co-culture technique for the production of PPT by P. hexandrum using hairy roots of L. flavum showed that PPT concentration was increased to 240% and 72% in dual shake flasks and dual bioreactors respectively (Lin et al., 2003). Transgenic hairy root cultures from L. mucronatum can also be used for scaling-up production of PPT. The maximum PPT amount obtained by transgenic hairy root was 5.78 mg/g DW (Samadi et al., 2014).