Transformation of Amorphadiene Synthase and Antisilencing P19 Genes into Artemisia annua L. and its Effect on Antimalarial Artemisinin Production

Purpose : The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens, and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads-p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads-p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads-p19 up to 2.57 folds compared to the untransformed leaves, while for p19, cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua.

Biological activity and hairy roots induction of Hibiscus sabdariffa L.

Hibiscus sabdariffa L. has been used traditionally in many countries of the world as food, especially as a flavouring agent in food industry. H. Sabdariffa is used for treating heart, nerve, liver disease, high blood pressure, arteriosclerosis, sore throat, cough, hypoglycaemia, laxative, diuretic, kidney stone, scurvy... The aims of this study are evaluation of bioactivities of H. Sabdariffa and the production of transformed hairy root of H. Sabdariffa for pharmaceutical production. In this study, Yen and Duh method showed the reducing power of ethanol the root extract and leaf extract are higher than that of stem. The root extract showed the α-glucosidase inhibitory activity with IC50 at 0.2 mg/mL is higher than those of stem and leaf. These results shows that root has higher bioactivites than stem and leaf. In this study, hairy roots of H. Sabdariffa were successfully induced via Agrobacterium rhizogenes ATCC 15834 in the plant cells. The frequency of hairy root and number of hairy root induction from the wounded sites of leaves are the highest (100% and 12.89 roots). The stable introduction of rolB and rolC genes of A. rhizogenes ATCC 15834 into H. Sabdariffa plants was confirmed by PCR analysis. Besides, the absence of virG gene confirmed hairy roots as bacteria-free. Subsequently, these results demonstrated that H. Sabdariffa, particularly the roots, has great potential as pharmacological values and hairy root production can be used as pharmaceutical sources.

Production of the Anticancer Compound Withaferin A from Genetically Transformed Hairy Root Cultures of Withania Somnifera

Withanolides (WTDs), well-known medicinally important compounds of Withania somnifera, including the anticancer compound withaferin A (WFA), are biosynthesized from their dedicated precursor squalene. Condensation of farnesyl pyrophosphate (FPP) molecules to produce squalene is catalyzed by squalene synthase (SQS). In the present study, the Arabidopsis thaliana squalene synthase gene ( AtSQS1) was transformed in W. somnifera by Agrobacterium tumefaciens C58C1 (pRiA4). The AtSQS1 gene was detected in 87.88 % of induced transformed hairy roots (THRs). The results showed that the growth index (GI) of THRs after five weeks of culture was 2-fold higher than that of adventitious hairy roots (AHRs). The biomass doubling time of THRs and AHRs was 18 and 30 days, respectively. Quantitative evaluation of WFA in the studied roots showed that THRs contain 1.51-fold more WFA (330±0.87μg g−1 dry weight (DW)) than AHRs (218±0.17μg g−1 DW). These findings can be used for the production of WFA as a valuable anticancer compound through controlled and scaled up cultures of W. somnifera THRs.

Improvement of Polyunsaturated Fatty Acid Production in Echium acanthocarpum Transformed Hairy Root Cultures by Application of Different Abiotic Stress Conditions

Fatty acids are of great nutritional, therapeutic, and physiological importance, especially the polyunsaturated n-3 fatty acids, possessing larger carbon chains and abundant double bonds or their immediate precursors. A few higher plant species are able to accumulate these compounds, like those belonging to the Echium genus. Here, the novel E. acanthocarpum hairy root system, which is able to accumulate many fatty acids, including stearidonic and α-linolenic acids, was optimized for a better production. The application of abiotic stress resulted in larger yields of stearidonic and α-linolenic acids, 60 and 35%, respectively, with a decrease in linoleic acid, when grown in a nutrient medium consisting of B5 basal salts, sucrose or glucose, and, more importantly, at a temperature of 15∘C. The application of osmotic stress employing sorbitol showed no positive influence on the fatty acid yields; furthermore, the combination of a lower culture temperature and glucose did not show a cumulative boosting effect on the yield, although this carbon source was similarly attractive. The abiotic stress also influenced the lipid profile of the cultures, significantly increasing the phosphatidylglycerol fraction but not the total lipid neither their biomass, proving the appropriateness of applying various abiotic stress in this culture to achieve larger yields.