Artemisia Annua Hairy Roots Research Articles

Heterologous expression of cyanobacterial PCS confers augmented arsenic and cadmium stress tolerance and higher artemisinin in Artemisia annua hairy roots.

The present study provides the first report of heterologous expression of phytochelatin synthase from Anabaena PCC 7120 (anaPCS) into the hairy roots of Artemisia annua. Transformed hairy roots of A. annua expressing anaPCS gene showed better tolerance to heavy metals, viz., arsenic (As) and cadmium (Cd) owing to 143 and 191% more As- and Cd-accumulation, respectively, as compared to normal roots with a bioconcentration factor (BCF) of 9.7 and 21.1 for As and Cd, respectively. Under As and Cd stresses, transformed hairy roots possessed significantly higher amounts of phytochelatins and thiols probably due to the presence of both AaPCS (Artemisia annua PCS) and anaPCS. In addition, artemisinin synthesis was also induced in transformed hairy roots under heavy metals stresses. In-silico analysis revealed the presence of conserved motifs in both AaPCS and anaPCS sequences as well as structural modelling of PCS functional domain was conducted. Interaction of AaPCS and anaPCS proteins with CdCl2 and sodium arsenate gene ontology analysis gave insights to anaPCS functioning in transformed hairy roots of A. annua. The study provides transformed hairy roots of A. annua as an efficient tool for effective phytoremediation with added advantages of artemisinin extraction from hairy roots used for phytoremediation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11816-021-00682-5.

Stimulation of Artemisinin Production in Artemisia annua Hairy Roots by Ag-SiO2 Core-shell Nanoparticles

Silver nanoparticles, exhibiting unusual properties of ionic ability associated with the particle suspension, have been extensively used as the anti-microbial agent in medicine. The present study addressed the elicitation potential of nanosilver particles for the enhancing plant secondary metabolite production. Ag-SiO2 core–shell nanoparticles (AgNPs) with an average size of 101.8 ± 8.9 nm were prepared as elicitor to the hairy root cultures of Artemisia annua. Artemisinin content in the cultures was increased from 1.67 mg/g dry wt to 2.86 mg/g dry wt by the stimulation of AgNPs at 900 mg/L for 3 d. AgNP elicitation was related to the released dissolved Ag+ and nanoparticulate nature of AgNPs introduced to the cultures. AgNP treatment induced oxidative stress (H2O2 production) resulting in lipid peroxidation (increased malonyldialdehyde (MDA) accumulation), enhanced activities of catalase (CAT) and increased artemisinin content in hairy roots. The elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 13.3 mg/L, a 3.9-fold increase over the control. These results suggest that Ag nanoparticles can be used as a novel effective elicitor in plant biotechnology for the production of plant secondary metabolites.

Study of Various Factors for Enhancement of Artemisinin in Artemisia Annua Hairy Roots

Artemisinin is an alternate anti-malarial drug which is widely used in the cure of multi-drug resistant Plasmodium falciparum malaria. In this study, enhancement of artemisinin content by using several elicitors and precursors was attempted initially by using one at a time (OVAT) approach. The most potent elicitor(s) & precursor were thereafter identified by the detailed analysis of the responses with respect to biomass and bioactive compound with respect to the concentrations of different effectors. Central Composite Design (CCD) was thereafter used to identify the antagonistic or synergistic effects of high and low concentrations of different precursors and elicitors. The maximum artemisinin content obtained was 3.45 mg/g on 15d by using the elicitor methyl jasmonate (40 µg/l) and the precursors casein acid hydrolysate (50 µg/l), sodium acetate (500 µg/l). Effect of addition time of elicitors and precursors on overall optimum biomass growth and artemisinin yield and productivity was also established.

Stimulation of artemisinin biosynthesis in Artemisia annua hairy roots by oligogalacturonides

The different fractions of oligogalacturonides (OGA) from polygalacturonic acid by pectinase hydrolysate have been partially purified using column chromatography of Sephadex G-10. The isolated fraction OGA2 (degree of polymerization, DP = 4.57) was found to stimulate the accumulation of artemisinin in Artemisia annua hairy roots. When hairy roots of 16-day old cultures were exposed to the OGA elicitor (60
g/mL) for 4 days, the maximum production of artemisinin reached 11.3 mg/L, a 55.2% increase over the control. OGA could induce H2O2 production in hairy root culture as one of early defense events. Moreover, the OGA-induced reactive oxygen species (ROS) were involved in stimulating the artemisinin biosynthesis in the hairy roots. This is the first report on the stimulation of artemisinin production in hairy roots by an oligogalacturonide elicitor.

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in Artemisia annua hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2'R,3R,3'E,4E,8E)-1-O-beta-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N (omega)-nitro-L-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-D-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 microM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.

Using an aerosol deposition model to increase hairy root growth in a mist reactor

Gas-phase reactors, including the mist reactor, have distinct advantages over liquid-phase reactors including the ability to manipulate the gas composition, to allow effective gas exchange in a densely growing biomass, and to affect secondary metabolite production. Mathematical modeling suggested that roots in a mist reactor are often too sparsely packed to capture mist particles efficiently and cannot, therefore, meet the nutrient demands required to maintain high growth rates. Indeed, growth rates of Artemisia annua hairy roots increased significantly when the initial packing density increased or when a higher sucrose concentration was used in the medium. Growth kinetics for 2, 4, and 6 days, however, showed a decrease or stationary growth rate after only 4 days for both 3 and 5% sucrose feeds. Residual medium analyses indicated that carbon was not exhausted, nor were any of the other major nutrients including phosphate. Increasing the mist duty cycle at constant carbon flux through the reactor reduced the growth rates slightly. In general, the aerosol deposition model correctly predicted how to optimize hairy root growth in the mist reactor.

The effect of phytohormones on growth and artemisinin production in Artemisia annua hairy roots

Few studies have focused on the effect of a broad range of phytohormones on growth and secondary metabolism of a single hairy root species. We measured growth, development, and production of the antimalarial drug, artemisinin, in Artemisia annua hairy roots in response to the five main hormones: auxins, cytokinins, ethylene, gibberellins (GA), and abscisic acid (ABA). Single roots grown in six-well plates in medium B5 with 0.01 mgl−1 (0.029 μM) GA3 produced the highest values overall in terms of the number of lateral roots, length of the primary root, lateral root tip density, total lateral root length, and total root length. When the total root lengths are compared, the best conditions for stimulating elongation appear to be: GA 0.01 mgl−1 (0.029μM)> ABA 1.0 mgl−1 (3.78μM)=GA 0.02 mgl−1 (0.058μM). Bulk yields of biomass were inversely proportional to the concentration of each hormone tested. All cultures provided with ABA yielded the highest amount of biomass. Both 6-benzylaminopurine and 2-isopentenyladenine inhibited root growth, however, only 2-isopentenyladenine stimulated artemisinin production, more than twice that of the B5 controls, and more than any other hormone studied. These results will prove useful in increasing hairy root growth and artemisinin production.

Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.

A comparative study of mist and bubble column reactors in the in vitro production of artemisinin

Artemisia annua hairy roots grown in nutrient mist reactors produced nearly three times as much artemisinin as roots grown in bubble column reactors, 2.64 µg/g DW and 0.98 µg/g DW, respectively.

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g−1 dry wt to 1 mg g−1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml−1 for 4 days, the maximum production of artemisinin reached to 13 mg l−1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.