AbstractsBiology & Animal Science

Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of 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 it was necessary first to develop different analytical methods to determine the alkaloid contents and changes in their profiles in transgenic cultures and after various treatments. Targeted and non-targeted analyses from cell andorgan cultures 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. Callus cultures were successfully initiated from five different explants onmodified B5 medium containing phytohormones. The phenotypes of the calli varied, but as was expected the callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems had elevated levels of strictosidine lactam compared to other cultures. Transformation experiments revealed that only leaves but not cotyledons, hypocotyls or stem segments were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. Wild type and gus hairy root clones contained twofold higher amounts of alkaloids than adventitious roots. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS analysis allowed the separation of the most volatile and non-polar alkaloids in a single run. The composition of typical non-polar alkaloids indicated the occurrence of 20 TIAs belonging to nine different groups. The quantities of these alkaloids varied between clones in the order eburenine, vincanine, vallesiachotamine and yohimbine isomer II. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GC-MS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Methyl jasmonate, a well-known elicitor, caused a significant increase in the total alkaloid content of wild type hairy roots as determined by NMR analyses. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased compared to non-elicited cultures. Another studied elicitor, chitosan, did not have any effect on individual alkaloid contents. Transgenic hairy root clones did not exhibit phenotype differences. Multivariate analysis from NMR data showed a clear discrimination…