## 4 Empirical Strategies for Improving TIA Production
### Empirical Optimization of the Culture Medium and Culture Conditions

Table 1: _Cont._
\begin{table}
\begin{tabular}{l l l l l} \hline
**Material** & **Strategy** & **Total TIAs** & **Observations** & **Refs** \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of & \multirow{2}{*}{ND} & 4-fold increase in hydhammericine & \multirow{2}{*}{[88]} \\  & _DAT_ & & (0.16 mg/g DW) & \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of & 1.5-fold increase & \multirow{2}{*}{5-fold increase in serpentine (3.7 mg/g DW) 3-fold increase in ajmalicine (0.35 mg/g DW) 4-fold increase in ajmalicine (0.35 mg/g DW) 5} \\  & CrPrx & (85 mg/g DW) & \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of & \multirow{2}{*}{NV} & 1.66-fold increase in ajmalicine & \multirow{2}{*}{[90]} \\  & _DXS_ & & (1.5 mg/g DW) & \\  & _DXS_ & & (1.66-fold increase in lochnericine & \multirow{2}{*}{[90]} \\  & _DXS_ & & (1 mg/g DW) & \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of & \multirow{2}{*}{NV} & 1.25-fold increase in lochnericine & \multirow{2}{*}{[91]} \\  & _ASa_ & & (2.5 mg/g DW) & \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of & 0.0072 mg/g & 1.35-fold increase in tabsorine & \multirow{2}{*}{[97]} \\  & _G10H/DXS_ & DW & (0.9 mg/g DW) & \\  & _G10H/DXS_ & DW & 1.15-fold increase in lochnericine & \multirow{2}{*}{[97]} \\  & & & (1.4 mg/g DW) & \\ \hline \multirow{2}{*}{Leaves} & \multirow{2}{*}{Overexpression of \(\mathit{ASa/DXS}\)} & 1.16-fold increase in tabsorine & \multirow{2}{*}{[91]} \\  & _OVER_ & (1.7 mg/g DW) & \\  & _ASa/DXS_ & DW & 1.18-fold increase in lochnericine & \multirow{2}{*}{[92]} \\  & & (2 mg/g DW) & \\ \hline \multirow{2}{*}{Leaves} & Transient & \multirow{2}{*}{ND} & \multirow{2}{*}{1.6-fold increase in vindoline (2.5 mg/g DW) 5} \\  & overexpression of & _GPPS_ & & \\ \hline \multirow{2}{*}{Tobacco cell cultures} & Overexpression of \(\mathit{TDC/STR}\) & \multirow{2}{*}{ND} & \multirow{2}{*}{Enhancement in strictosidine (5.3 mg/L) 5} & \multirow{2}{*}{[93]} \\  & _MYR_ & (1.7 mg/g DW) & \\ \hline \multirow{2}{*}{Hairy root culture} & Overexpression of \(\mathit{TDC/STR}\) & \multirow{2}{*}{ND} & \multirow{2}{*}{Enhancement in strictosidine (21.2 mg/L) 5} & \multirow{2}{*}{[93]} \\  & _MYR_ & (1.95 mg/g FW) & \\ \hline \multirow{2}{*}{_Saccharomycesces cerevisiae_} & Overexpression of \(\mathit{STR/SGD}\) & \multirow{2}{*}{ND} & \multirow{2}{*}{Enhancement in strictosidine (2000 mg/L) 5} & \multirow{2}{*}{[94]} \\  & Overexpression of \(\mathit{TDC/STR}\) & \multirow{2}{*}{ND} & \multirow{2}{*}{3-fold increase in serpentine (0.291 mg/g DW) 5} & \multirow{2}{*}{[95]} \\  & _MYRKY1_ & & (0.015 mg/g DW) & \\ \hline \multirow{2}{*}{Leaves} & \multirow{2}{*}{Transient \(\mathit{overexpression of  
As regards the effect of plant growth regulators, 2,4-dichlorophenoxyayacetic acid inhibited TIA production [100; 101], even when the precursors, loganin and tryptamine, are added to _C. roseus_ cell cultures [76]. Similarly, the addition of ethylene inhibited the production of ajmalicine [102]. However, the combination of benzyladenine or kinetin with indole-3-acetic acid led to an enhancement of TIA production (total alkaloid yield of over 45 mg/L). Moreover, [PERSON] _et al._[74] found high levels of ajmalicine (over 0.85 g/L) released in the spent media of _C. roseus_ shoot grown in the presence of a high level of benzyladenine (8.90 mM) and a low level of indole-3-acetic acid (2.85 mM) (Table 1), whereas the addition of a high concentration of indole-3-acetic acid (11.42 mM) and a low concentration of benzyladenine (2.22 mM) resulted in high levels of ajmalicine (over 0.40 g/L) being accumulated in the shoots. In addition, TIA biosynthesis improved when _C. roseus_ callus were treated with benzyladenine and indole-3-acetic acid and exposed to the light, especially vindoline (0.19 mg/g DW) and serpentine (0.53 mg/g DW) compared with callus that was not exposed to light [101].