## 4 Empirical Strategies for Improving TIA Production
### Empirical Optimization of the Culture Medium and Culture Conditions
On the other hand, [PERSON] and [PERSON] [75] showed that different O\({}_{2}\) concentration decreased both cell growth and ajmalicine biosynthesis in _C. roseus_ immobilized cells. However, the ajmalicine biosynthesis was unaffected when a high concentration of O\({}_{2}\) and CO\({}_{2}\) was used. Feeding with Precursors and Elicitation as Empirical Strategies for Increasing the Production of TIAs  
Sometimes, feeding with specific precursors has proved to be a successful strategy to increase the levels of TIAs using _in vitro_ cultures. Specifically, tryptophane, tryptamine, geraniol, 10-hydroxygeraniol, loganin and secologanin have been the precursors most extensively added [65; 76; 77; 100; 103; 104; 105]. In fact, the combined use of tryptamine and loganin improved the accumulation of ajmalicine [100] and provoked a high accumulation of strictosidine [76] in _C. roseus_ cell cultures. Also, the addition of succinic acid, tryptamine and tryptophan to the culture medium significantly increased ajmalicine and catharanthine levels [65] (Table 1). Similarly, the levels of ajmalicine and strictosidine were enhanced when _C. roseus_ cell cultures were fed with loganic acid, loganin and secologanin [105]. More recently, \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Material** & **Strategy** & **Total TIAs** & **Observations** & **Refs** \\ \hline \multirow{8}{*}{Transgenic plant} & & & 3.03-fold increase in vindoline \\  & & & (2.1 mg/g DW) \\  & & & 2.29-fold increase in catharanthine \\  & & & (4.6 mg/g DW) \\  & \multirow{2}{*}{ND} & 6.30-fold increase in ajmalicine \\  & & & (0.315 mg/g DW) \\  & & & 1.08-fold increase in anhydrovinblastine \\  & & & (10.2 mg/g DW) \\  & & & 10.2-fold increase in vinblastine \\  & & & (0.27 mg/g DW) \\ \hline \hline \end{tabular}  
* Abbreviations: DW, dry weight; FW, fresh weight; ND, not determined; NV, no variation; TIAs, Terpenoid indole alkaloids; STR, strictosidine synthase; TDC, tryptophan decarboxylase; DAT, deacetylulroline 4-O-acetyltransferase; CrPrx, _C. roseus_ peroxidase; DXS, 1-deoxy-D-xylulose-synthae; G10H, geraniol-10-hydroxy; ASa, anthranilate synthase \(a\) subunit; GPPS, Geranyl diphosphate synthase; CrMPK3, _C. roseus_ mitogen activated protein kinase 3, MeJA, methyl jasmonate; CDs, cyclodext; BA, benzyl adenine; IAA, indole-3-acetic acid.  
\end{table} 
Table 1: _Cont._feeding experiments with geraniol (0.5 mM), hydroxygeraniol or loganin provoked a marked increase in the accumulation of tabersonine (around 1.4 mg/g DW) in _C. roseus_ hairy root cultures [77] (Table 1), but the most spectacular results were found in loganin feeding experiments where TIA accumulation improved significantly compared to non-transformed cells [104]. On the other hand, elicitation is one of the most effective established strategies to improve secondary metabolite production in plant platforms [106]. Both abiotic and biotic elicitors have been extensively used for improving TIA production in _C. roseus in vitro_ cultures since the beginning of the 1990s [105, 107, 108, 109]. In fact, abiotic elicitors like metal ions and UV light, as well as biotic elicitors such as fungal preparations and yeast extracts, hydrolytic enzymes, chemicals (cyclodextrintrins (CDs) and signalling molecules like nitric oxide, jasmonic acid and derived compounds such as methyl jasmonate (MeJA) and acetylsalicylic acid were commonly used [73, 84, 85, 110, 111, 112]. In this way, the treatment of _C. roseus_ hairy root cultures with 0.1 mM sodium nitroprusside (NO donor) caused a significant increase of serpentine, catharanthine, ajmalicine, horn hammericine, lochnericine and tabersonine [78] (Table 1). Also working with hairy roots of _C. roseus_ treated with 101.9 \(\upmu\)M of jasmonic acid, [PERSON] _et al._[79] showed an increase of 190% in tabersonine (Table 1). In the same way, [PERSON] _et al._[110] showed that the addition of 100 \(\upmu\)M MeJA to _C. roseus_ cell cultures increased the ajmalicine extracellular production (10.2 mg/L) in a dose and time-dependent manner.