TRANSIENT TRANSFORMATION OF Artemisia annua L. LEAVES WITH ads, cyp71av1, and dbr2 GENES FOR ENHANCING ARTEMISININ CONTENT

• Main Author: Maharani, Harmita
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45139
• Institution: Institut Teknologi Bandung

Artemisinin is a sesquiterpene lactone used as antimalarial in artemisinin-based combination therapy (ACT). It is naturally produced in a small amount (0,01-1%) in the Artemisia annua L. plants. The effort is needed to enhance artemisinin content in order to support its role as antimalarial drug. The objective of the research is to increase artemisinin content in A. annua leaves through the transient transformation of ads (amorpha-4,11-diene synthase), cyp71av1 (cytochrome P450 monooxygenase), and dbr2 (double bond reductase) gene mediated by Agrobacterium tumefaciens. The third to seventh leaves were transformed by using infiltration vacuum method. Transgenes were confirmed based on the blue area of histochemical GUS assay and DNA genome isolated from transformant leaves, and metabolite content was measured using HPLC (high-performance liquid chromatography). Transformation efficiency of ads, cyp71av1, dbr2, and co-transformation efficiency of ads and cyp71av1, ads and dbr2, cyp71av1 and dbr2, and all of genes were 32,44%, 2,49%, 0%, 56,34%, 45,06%, 0,02%, and 33,05%, respectively. Control nontransformed leaves showed 0,013% (w/w) artemisinin content. Leaves transformed by a single recombinant plasmid of ads, cyp71av1, and dbr2 showed 0,013%, 0,020%, and 0,022% (w/w) artemisinin content, respectively. Leaves transformed by double (co-transformation) recombinant plasmids consisted of ads and cyp71av1, ads and dbr2, cyp71av1 and dbr2, and all of genes showed 0,010%, 0,005%, 0,013%, and 0,005% (w/w) artemisinin content, respectively. Leaves transformed by a single recombinant plasmid of dbr2 showed the highest artemisinin content compared to other recombinant plasmids treatment, that was 1,6-fold higher than control.