## 4 Empirical Strategies for Improving TIA Production
### Empirical Optimization of the Culture Medium and Culture Conditions
MeJA also triggered the production of ajmalicine (137.2 mg/L) and catharanthine (55.6 mg/L) in _C. roseus_ cell cultures [73]. This elicitor was also effective in accumulating ajmalicine (6.34 mg/g DW) serpentine (1.71 mg/g DW), ajmaline (12 mg/g DW) and catharanthine (4.34 mg/g DW) in _C. roseus_ hairy roots elicited with 250 \(\upmu\)M MeJA [81] (Table 1). Likewise, the addition of 1 mM of acetylsalicylic acid provoked an increase of 505% total alkaloids in _C. roseus_ cells transformed with _Agrobacteriumum tumefaciens_[113]. Several studies carried out with fungal preparations have proved to be a successful approach to increase TIA production in _C. roseus in vitro_ cultures. Indeed, a maximum yield of ajmalicine (166 \(\upmu\)g/g DW) was obtained from 20-d old cell cultures treated with _Trichoderma viride_[82]. In addition, the elicitation with a fungal preparation derived from _Penicilium citrinum_ or PB90 (a protein elicitor from _Phytophthora boehmeriae_) also increased the production of catharanthine reaching levels over 20 mg/L in both cases in _C. roseus_ cell cultures [83, 111] (Table 1). Another class of compounds that are gaining attention as potential elicitors of secondary metabolism in plants are cyclodext (CDs) which are modified cyclic oligosaccharides derived from starch. CDs have been able to induce defence responses in _C. roseus_ cell cultures enhancing ajmalicine (over 200 mg/L) and catharanthine (85 mg/L) levels [73, 80] (Table 1). Abiotic elicitors including UV-light or heavy metals also induce TIA production in _C. roseus_. Thus, hairy roots exposed to UV-B increased significantly the levels of lochnericine (over 2.7 mg/g DW), serpentine (over 0.62 mg/g DW), and ajmalicine (over 0.31 mg/g DW) [114]. [PERSON] and [PERSON] [84] also observed an enhancement of catharanthine (0.12 mg/g DW) and vindoline (0.06 mg/g DW) when _C. roseus_ cell cultures were exposed to UV-B light. Moreover, a short UV-C irradiation promoted the production of 90 mg/L ajmalicine and 50 mg/L catharanthine in _C. roseus_ cell cultures [73] (Table 1). In relation to heavy metals, the treatment of 50 uM chromium raised vincristine and vinblastine content in _C. roseus_ plants, particularly in shoots reaching levels of around 2 and 2.25 ug/g DW, respectively [35] (Table 1). Biotic and abiotic elicitors are associated to different mechanisms of elicitation and, when used in combination, could enhance metabolite accumulation in plant cell cultures. Indeed, the joint action of _Aspergillus niger_ mycelium and tetramethylammonium bromide induced a synergism on TIA accumulation in _C. roseus_ cell cultures, since they increased the levels of ajmalicine (63 mg/L) and catharanthine (17 mg/L). Also, the combination of malate and sodium alginate resulted in a high ajmalicine (41 mg/L) and catharanthine (26 mg/L) accumulation [65,68] (Table 1). A new strategy for increasing TIA production in _C. roseus_ cell cultures is the combined use of CDs and MeJA. In fact, when _C. roseus_ cell cultures were elicited with both elicitors a high accumulation of ajmalicine and catharanthine was detected (450 and 155 mg/L, respectively). This effect was even greater when these elicitors were combined with short UV light exposure reaching a production of ajmalicine and catharanthine of 1040 mg/L (85 mg/g DW) and 196 mg/L (10 mg/g DW), respectively [73] (Table 1).