Alkaloids of in vitro cultures of Rhazya stricta:Dissertation

Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in Middle East and South Asia. It contains a large number of terpenoid indole alkaloids (TIAs), some of which possess interesting pharmacological properties. This study was focused on biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities, therefore, many analytical methods were developed to investigate the alkaloid contents in in vitro cultures. Targeted and non-targeted analyses were carried out using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems contained elevated levels of strictosidine lactam compared to other cultures. The results of transformation experiments revealed that only leaves were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS indicated the occurrence of 20 non-polar TIAs belonging to nine different groups. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GCMS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased as a result of elicitation by methyl jasmonate (MeJA). Multivariate analysis of NMR data showed a clear discrimination between transformed and control cultures. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabling comprehensive investigation of the alkaloids. These data serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering