TRANSFORMATION OF DBR2 AND P19 GENES IN ARTEMISIA ANNUA L. PLANT TISSUE CULTURE VIA AGROBACTERIUM TUMEFACIENS MEDIATION AND METABOLITES ANALYSIS

• Main Author: Sy, Khairunnisa
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76432
• Institution: Institut Teknologi Bandung

Background and purpose: Malaria is still a globally threatening disease that affects 247 million people and causes 619,000 deaths. Nowadays, anti-malarial therapy relies on artemisinin compounds that are mostly produced from native plants. The diminished levels of artemisinin in Artemisia annua L., which are associated with the biosynthetic pathway, are influenced by specific enzymes involved in its formation. Genetic engineering offers a potential solution to increase the content of artemisinin by regulating the genes involved in its biosynthesis. The objective of this study is to introduce the DBR2 and P19 genes as anti-silencing agents into A. annua L. by Agrobacterium-mediated transformation. Then, assess their impact on artemisinin and its precursor production, such as artemisinic acid (AA) and dihidroartemisinic acid (DHAA). Methods: The existence of DBR2 and P19 genes was verified by analyzing polymerase chain reaction (PCR) products. Plasmids carrying DBR2 and P19 genes were transformed into A. tumefaciens AGL1, and subsequently introduced into the leaves from plant tissue culture of A. annua L. which came from sterile seed germination, using vacuum infiltration techniques. The effectiveness of the transformation was assessed through the GUS histochemical assay. The instrument of Ultra-performance liquid chromatography iv (UPLC)-ESI-MS/MS was employed to measure the levels of artemisinin, artemisinic acid (AA), and dihydroartemisinic acid (DHAA). Results: The gene of DBR2 and P19 were confirmed in plasmid pCAMBIA1303. The transformation in A. tumefaciens AGL1 was successful using a freeze-thaw method and confirmed by PCR. The plant tissue culture from seeds of A. annua, L., has been available for infection by A. tumefaciens using vacuum infiltration. Based on GUS histochemical assay, the gene has been successful to insert in A. annua L. leaves with an efficiency of 48.2%. The evaluation result by UPLC-ESI-MS/MS showed that the level of artemisinin in the transformation sample with pCAMBIA-dbr2-p19, AGL1 without plasmid, was detected but not quantifiable. The level of artemisinin in wild type leaves was detected and quantifiable with a result of 0.008% in fresh weight. For the artemisinic acid (AA) and dihydroartemisinic acid (DHAA) precursors, were not detectable in the transformation sample. However, they were only detectable in the wild type. These precursors might have been lost during the co-cultivation process which took 3 days. Conclusion: DBR2 and the anti-silencing P19 gene could be inserted into A. tumefaciens AGL1 and the transformation to A. annua L was successful. The level of artemisinin was detected but the level of artemisinic acid and dihydroartemisinic acid was not detected.